Accrington Stanley knocked out League 1 Tranmere Rovers, as they went through 5-3 on penalties at Prenton Park.
Ray Putterill gave the visitors the lead on 55 minutes but Enoch Showunmi levelled up for Les Parry's side to send the game to penalties, which Stanley came through unscathed.
League 2 side Port Vale knocked out Rochdale, who were promoted to League 1 in May.
Marc Richards opened the scoring at Vale Park, on 27 minutes, before his namesake Justin added a second twenty minutes before the end. Matt Done pulled a goal back for Keith Hill's side but it was all too little too late.
Another League 1 side crashed out to League 2 opposition at the Banks Stadium as Chesterfield beat Walsall 2-1.
The Spireites took the lead with just 13 minutes on the clock, when Jordan Bowery scored his first of the season. Walsall were back level six minutes later through Reuben Reid but Dean Morgan then secured the victory with his first goal for the club, since signing earlier this month.
Shrewsbury Town kept up their excellent form by knocking out Oldham Athletic at Boundary Park.
Steven Leslie scored the only goal of the game seven minutes from time to earn his side a place in Round 2.
Torquay United also continued their fantastic start with a win over Bournemouth at Dean Court.
Neither side could break the deadlock during the 90 minutes so the game went to penalties, with the Gulls winning 3-0.
SAINTS DON'T GO MARCHING ON
Holders Southampton crashed out of the competition at the first attempt as they were beaten 3-0 at home by Swindon Town.
Two goals from Vincent Pericard, came either side of a strike by Charlie Austin to secure the Robins' route into the next round and leave the Saints caretaker boss Dean Wilkins looking for his first win.
LONG TRIP HOME
Northampton Town face a long trip south after crashing out of the Johnstone's Paint Trophy away at Hartlepool United.
The Pools, still reeling from Saturday's 5-0 drubbing at the hands of Sheffield Wednesday, bounced back in style, with a 4-0 win at Victoria Park.
All four goals came in a nine minute period of the second half, starting on 66 minutes through Evan Horwood. Antony Sweeney made it 2-0 three minutes later before Andy Monkhouse and Denis Behan wrapped up the scoring.