Juventud Unida Universitario have stunned Union Santa Fe in their Copa Argentina second round clash on Wednesday.
The third-tier outfit held firm at their Estadio Provincial Juan Gilberto Funes home, finishing level at 0-0 before winning the penalty shootout 4-1 against Union Santa Fe.
Union Santa Fe, who have performed well in the second tier, were expected to progress but were always in danger once the hosts forced a shootout.
That proved to be the only upset on Wednesday as two other second-tier outfits advanced.
San Martin San Juan managed to get the job done in regulation, claiming a 2-1 home win against third-tier Cipolletti.
Gabriel Chironi put the underdogs ahead on 37 minutes but the hosts levelled early in the second half.
Lucas Salas struck a dramatic late winner just a minute before full-time for San Martin San Juan.
The clash between Patronato and Instituto finished 2-2 before the latter claimed a thrilling 5-4 shootout win.
Instituto had twice come from behind and made the most of doing so by clinching the shootout victory.