Defending champions Real Madrid suffered a 2-1 defeat at Real Betis on Saturday, which coach Carlo Ancelotti said was a "big blow" to their title defence, while Atletico Madrid took advantage to go above them in the table.
For Betis, Isco scored the winner from the penalty spot against his former side. Ancelotti picked a strong line-up, with no breathing room in the title race, despite the impending Champions League clash against rivals Atletico in midweek.
"If we play like this on Tuesday we will not win," said Ancelotti, whose side have just 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 Madrid 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 Madrid before departing in 2022.
Madrid 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, Madrid were outfought by a determined 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."
Meanwhile, Atletico Madrid held their nerve at the Metropolitano stadium to claim an important three points. After substitute Julian Alvarez struck, Athletic Club hit the woodwork three times in the hunt for an equaliser.
"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.
"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.
