Former FA Cup winner Coventry City was dumped out of the competition after losing 2-1 to Conference North side Worcester City.
League One Coventry sits 64 places above Worcester in the English football pyramid, but it was undone by its non-league opposition, which made the most of having a numerical advantage for 51 minutes.
Coventry goalkeeper Lee Burge was dismissed six minutes before the break after an altercation with Daniel Nti and Sean Geddes converted the resulting spot-kick to give the visitor the lead.
The host was given a lifeline when James Maddison won a debatable penalty, but Reda Johnson saw his effort saved by Nathan Vaughan and Worcester capitalised just after the break.
Geddes found the net for a second time with a fine low drive from just inside the area and, although Johnson made amends for his earlier miss nine minutes from time, Worcester held on to send the 1987 cup winner out of the Cup.
Portsmouth, a winner in 2008 and a finalist in 2010, was held to a 2-2 home draw by Conference National side Aldershot.
The host took the lead through Jed Wallace's 16th-minute penalty, but it was pegged back by goals from Jordan Roberts and Mark Molesley.
Danny Hollands ultimately spared Portsmouth's blushes in the 81st minute and, although Paul Robinson was dismissed a few moments later, the host held on to force a replay.
The highest scoring contest of the round saw Colchester United defeat host Gosport Borough 6-3, while Chesterfield also hit six on the road, beating Braintree Town 6-0.
Norton United was the lowest ranked team in action and it was ultimately thumped 4-0 by Rob Ramshaw-inspired Gateshead, with the forward scoring three times.
Bradford City came from behind to beat Halifax Town 2-1 in the day's early match, sparing itself from an embarrassing exit against its Yorkshire rival, while Altrincham lost 4-1 to Blyth Spartans, and Wrexham beat Woking 3-0.
Scunthorpe United won 2-0 at Forest Green Rovers, while Maidstone United and Accrington Stanley held Stevenage and Notts County respectively in the only goalless games of the day.