The third-tier Cipolletti upset a side in the division above them – Villa San Carlos – 4-2 on penalties after their clash finished locked at 1-1.
Santiago Vergara had given visitors Cipolletti a 77th-minute lead with a spot-kick, but his side were reduced to 10 men moments later when Lucas Mellado saw red.
Substitute Diego Mendoza struck from the penalty spot for the hosts five minutes later and a shootout followed.
And Cipolletti got the better of their more-fancied rivals in the spot-kicks to claim an upset victory.
Elsewhere, second-tier Brown de Adrogue battled past fourth-tier Villa Cubas 3-2.
Juan Manuel Garcia netted a brace for the winners, who were never behind and held on at the Estadio Bicentenario Ciudad de Catamarca.
Second-tier Instituto got past Chacarita Juniors 3-1 and Crucero del Norte were 2-0 victors at home to Gimnasia Concepcion thanks to Victor Aquino's brace.
