LaLiga champions Real Madrid suffered a 2-1 defeat at Real Betis Saturday, which coach Carlo Ancelotti said was a "big blow" to their title defence, while Atletico Madrid took advantage to move top of the table.
Diego Simeone's side scraped a nervy 1-0 win against Athletic Bilbao, fourth, to lead Barcelona and Real by two points.
For Betis, Isco scored the winner from the penalty spot against his former side Real, third, with Barca in action Sunday against Real Sociedad and able to reclaim top spot with a win.
Real coach Carlo Ancelotti picked a strong line up, with no breathing room in the title race, despite the impending Champions League clash against rivals Atletico.
"If we play like this Tuesday we will not win," said Ancelotti, whose side have one win in their last five Liga matches.
The visitors took the lead at the Benito Villamarin stadium through Brahim Diaz but Betis struck back with a Johnny Cardoso header, as Real missed the suspended Jude Bellingham.
Isco secured the Andalusian side a deserved victory with a penalty early in the second half.
"It's a big blow. We have to react," continued Ancelotti.
"We started well, but later we did not have the same level of commitment, the game got out of control against a side that played better than us and deserved to win."
Isco returned to action in December after missing several months with a broken leg and has hit top form in recent weeks.
"About halfway through the first half we stepped up our game, and I'm happy with the win against the best team around these days," said the playmaker, who won several major trophies with Real before departing in 2022.
Real started the stronger and opened the scoring after just 10 minutes, putting together a fine team move with 19 passes.
It culminated when Kylian Mbappe found Ferland Mendy, who squared for Diaz to tuck home.
Betis soon found their footing and Cardoso powered a header past Thibaut Courtois from Isco's corner to level.
Without Bellingham's dynamism in midfield Real were outfought by Betis.
Manuel Pellegrini's side took the lead early in the second half from the penalty spot when Antonio Rudiger crashed into Jesus Rodriguez as he ran in on goal.
Isco smashed home for his sixth goal across all competitions.
For the final 15 minutes, Ancelotti replaced Mbappe, who had dental trouble ahead of the game, with Endrick.
"He had that (tooth) problem, he hadn't trained a lot," explained Ancelotti.
"He was not at his best level."
Atletico held their nerve at the Metropolitano stadium to take a provisional lead in the title race.
After substitute Alvarez struck, the Basque side hit the woodwork three times in the hunt for an equalizer.
"It's an incredibly important win, we knew it would not be easy," Alvarez told Movistar.
"We're in the fight for the title, but we know there's a long way to go, a lot of very important games are coming close together, but with hard work and humility we'll achieve (our goals)."
Atletico goalkeeper Jan Oblak made a fine save to deny Inaki Williams early on, in an even first half.
The Rojiblancos have often relied on their substitutes making huge impacts this season and so it proved again as they opened the scoring.
Simeone brought on Marcos Llorente and Alvarez just before the hour mark and the duo combined to open the scoring after 66 minutes.
Llorente delivered a fine low pass for the former Manchester City striker to run on to, and he finished with minimal fuss for his eighth league goal of the campaign.
Athletic came millimeters away from an equalizer when Benat Prados headed against the post with Oblak beaten and Williams crashed the rebound off the underside of the crossbar and out.
"In the second half our changes helped us, Oblak too, and the goddess of fortune with the shots that hit the post and the bar," said Atletico coach Simeone.
Ghana international Williams hit the woodwork once again with a deflected strike from range and Athletic left frustrated.
"I think we deserved a lot more, we had more chances than them, but it's not about who deserves what, it's about putting them away," Williams told Movistar.
RESULTS
Girona 2 (Tsygankov 21, Herrera 68) Celta 2 (Losada 36, Alonso 51-P)
Vallecano 1 (Ratiu 55) Sevilla 1 (Lukebakio 81)
Betis 2 (Cardoso 34, Isco 54-P) Real Madrid 1 (Diaz 10)
Atletico 1 (Alvarez 66) Athletic Club 0
Friday
Valladolid 1 (Latasa 63) Las Palmas 1 (Sandro 22)