Australia declared their first innings at 565 for five before striking early in South Africa's second innings as they seek an unlikely victory in the first Test.
Skipper Michael Clarke called a halt to Australia's huge first innings with Australia 115 runs ahead as he sought to put the world's number one team under pressure.
The move paid immediate dividends with opener Alviro Petersen dismissed for five in the second over, leaving the tourists effectively two wickets down with JP Duminy unable to bat after suffering a series-ending Achilles injury.
Australia also thought they had first-innings centurion Hashim Amla out when he chopped onto his stumps only for a review to show that James Pattinson had over-stepped for a no-ball, giving Amla a reprieve.
At lunch, South Africa had steadied at 31 for one, with skipper Graeme Smith on 17 and Amla not out seven.
Clarke earlier passed up the opportunity of another triple century when he made the declaration, leaving him unbeaten on 259 with wicketkeeper Matthew Wade 19 not out.
Clarke eclipsed England's Alastair Cook's 235 not out two years ago as the highest individual score at the Gabba. Don Bradman hit 226 at the ground in 1931.
Australia went after quick runs before the declaration. Michael Hussey had a nervy spell before clinching his 17th Test century and was out on the next ball he faced.
The senior left-hander first survived an lbw appeal from Vernon Philander on 99 after the TV umpire found the ball had first nicked his bat.
Hussey then scrambled through for a single for his hundred and almost ran out batting partner Clarke in the process. He was out to a diving catch by fielding substitute Faf du Plessis at short cover off Morne Morkel.
Hussey's 129-ball innings was the fifth century of the Gabba Test and he shared in a 228-run stand for the fifth wicket with Clarke on the batting-friendly pitch.