After River Plate had moved level at the top with a victory over leaders Danubio on Saturday, a win for Nacional over reigning champions Penarol would have moved them a point clear at the summit.
However, the hosts were behind after 15 minutes when Jonathan Rodriguez opened the scoring and although Ivan Alonso equalised 12 minutes later, Penarol led at the break courtesy of Luis Aguiar.
Rodriguez completed his brace a minute after the interval and even though Diego Arismendi pulled one back six minutes from time, it was not enough for Nacional.
That meant Danubio remained top of the standings, despite being edged out 2-1 by River Plate at the Estadio Jardines del Hipodromo on Saturday.
The sides are now separated only by goal difference after River made a superb start, blowing away Danubio with two goals in the opening 11 minutes from Hamilton Pereira and Lucas Olaza.
Cristian Gonzalez's own goal three minutes after the hour made life uncomfortable for the visitors, but they held on to inflict a second league defeat in four matches on Danubio.
Meanwhile, Rentistas and Defensor Sporting played out an amazing 11-goal encounter at the Complejo Deportivo Rentistas, with the hosts running out 6-5 winners after Felipe Conceicao and Gonzalo Bazallo had opened the scoring with a goal for each side.
Adrian Luna and Anibal Hernandez then put the visitors 3-1 up within half an hour, before Gabriel Costa pulled a goal back for Rentistas.
The goals kept on coming as Luna completed his brace on the stroke of half-time, only for Marcelo Fernandez to reply, with Mario Risso restoring the two-goal lead for Defensor soon afterwards.
However, David Terans scored twice in four minutes before Fernandez grabbed a dramatic second in injury time to settle a pulsating contest, which also saw Facundo Moreira dismissed a minute from time.
Elsewhere, Wanderers' surge up the table continued, as they won their fourth league game in a row thanks to a Sergio Blanco penalty against Sud America.
Fenix's miserable form showed no signs of abating as they slipped to the bottom of the table thanks to a Jonathan Barboza effort after 39 minutes for Liverpool, while Juventud de Las Piedras' 2-2 draw against Cerro sees them in 15th.
El Tanque Sisley and Racing shared the points in a four-goal thriller, with Juan Pablo Fagundez atoning for his earlier own goal by registering an 84th-minute equaliser for El Tanque.
Federico Ramos picked the ideal time to score his first league goal of the season, striking in the 90th minute for Miramar Misiones to secure a 2-1 win over Cerro Largo.
