Barnsley lost 5-4 on penalties after a 0-0 draw at League One Bolton Wanderers in the EFL Cup first round on Tuesday evening.
Substitute Devante Cole was the only player to miss in the shoot-out on his 'second debut' for the club after a 2014 loan spell.
The Reds were on top in the first half but missed big chances then the hosts were the better team after the break.
Markus Schopp made eight changes from Saturday's draw at Cardiff City in the Championship opener, with only Michal Helik, Callum Styles and Cauley Woodrow staying in the side. There was no involvement at all for Callum Brittain, Liam Kitching, Ben Williams, Romal Palmer and Carlton Morris.
George Miller made his full debut – 922 days after signing for the club – at right wing-back, as did Josh Benson in central midfield. Herbie Kane and Devante Cole were on the bench after injuries as was teenager Jason Sraha.
Barnsley had a goal ruled ruled out in the third minute when Miller sent in an excellent cross to Victor Adeboyejo whose header was straight at the goalkeeper then Woodrow turned in from almost on the line but was flagged offside. Replays showed he was onside.
Clarke Oduor should have scored in the ninth minute when he met a left-wing Jordan Williams cross unmarked five yards out but scuffed the shot and the goalkeeper gathered.
Barnsley continued to dominate the ball but the match became more of an even contest, with Bolton's Antoni Sarcevic sending a long-range effort just wide of the top right corner.
Adeboyejo came close again on 29 minutes when he fired wide from the edge of the box on the right after Jasper Moon won the ball and Oduor played a good throughball.
Bolton could have taken the lead three minutes later with their first real chance. Sarcevic ran down the right of the Reds box, after a home corner was cleared, and passed to George Johnston whose low ten-yard shot was saved by Jack Walton diving down to his right.
Early in the second half, Ricardo Santos headed well wide from a right-wing corner by ex-Red Lloyd Isrove then another former Barnsley man Gethin Jones saw his effort from the right of the box beaten away by Walton.
Schopp brought on Cameron Thompson for his professional debut, in place of Adeboyejo, then replaced Styles and Woodrow with Kane and Cole.
Barnsley struggled to find the same rhythm in the second half but came close on 71 minutes when Cole headed a Miller cross at the goalkeeper under pressure from a defender 12 yards out.
Bolton almost won it in the 90th minute but Walton saved a Josh Sheehan shot from the edge of the box then Amadou Bakayoko headed the rebound over.
Benson, Kane, Helik and Jordan Williams all netted for Barnsley before Cole saw his effort saved by Dixon diving to his left and Bolton completed a perfect set of five spot-kicks to reach the second round.
Bolton (4-3-3): Dixon; Jones, Santos, Johnston, Gordon; MJ Williams, Lee (Sheehan 79mins), Sarcevic; Isgrove, Doyle (Kachunga 66), Delfouneso (Bakayoko 56). Unused: Sheehan, Thomason, Tutte, Brockbank, Darcy. Yellow cards: Jones.
Barnsley (3-4-3): Walton; Moon, Halme, Helik; Miller, Styles (Kane 60mins), Benson, J Williams; Oduor, Adeboyejo (Thompson 56), Woodrow (Cole 60).
Subs: Collins, Sraha, Sibbick, Frieser. Yellow cards: Benson, Kane.