Captain Pat Cummins confirmed at the toss that Scott Boland would come into the side for Lyon in the second Test, before rain returned to Gqeberha and the start was delayed.
Lyon has been overlooked twice when fit for pink-ball encounters in the past 18 months, but has played every daytime Test for Australia since July 2013.
But this is a significant change, with more grass left on a St George's Park wicket that is generally considered to be slow and friendly to spin.
Lyon has now missed five of the past 10 Tests for Australia through a combination of injury and selection, and bowled just two overs in Perth last summer.
The 38-year-old admitted he was "filthy" when left out of Australia's team against England for the day-night Test at the Gabba last year.
"(It's) partly conditions, and Scotty Boland has been incredible whenever he's played Test cricket," Cummins said at the toss.
"So it felt like it aligned this week and it was the right time to insert him.
"When there's anything in the wicket, he gets a lot of seam movement. He's super accurate, and even if it does flatten out he brings that control."
The signs Lyon wasn't playing were there on Sunday morning when he picked up a football during warm up, and appeared to congratulate Boland for his selection.
Australia also have the advantage of Matt Renshaw's off spin after his two wickets in the first Test win, along with Travis Head's tweakers and Beau Webster's ability to bowl off-breaks.
Lyon's omission was the only change for Australia, with Nic Maddinson to remain at the top of the order and Renshaw No.5 after his first Test heroics.
South Africa have welcomed back pace spearhead Kagiso Rabada in place of spinner Keshav Maharaj, with fellow tweaker Simon Harmer holding his spot.
The hosts produced one shock, with Marques Ackerman asked to open on debut after never doing so in his first-class career and Ryan Rickelton moving to No.6.
Matthew Breetzke, who has opened before, has also come into the side with Tristan Stubbs and Kyle Verreynne both axed. Rickelton will take the gloves from Verreynne.
Light rain had persisted across Gqeberha on Sunday morning, before a brief break allowed the toss.
More rain is forecast across the opening two days of the Test, although the heaviest is predicted overnight.
A win at St George's Park will wrap up the series for Australia after their 214-run victory at Kingsmead Stadium last week, with the third Test in Cape Town next week.
"Any away series you want to win," Cummins said.
"They're not easy to come by, so we're in a good position, one-nil up, so we're going to give this one our best shot."
AUSTRALIA: Travis Head, Nic Maddinson, Marnus Labuschagne, Steve Smith, Matt Renshaw, Alex Carey, Beau Webster, Mitchell Starc, Pat Cummins (c), Scott Boland, Josh Hazlewood.
SOUTH AFRICA: Aiden Markram, Marques Ackerman, David Bedingham, Temba Bavuma (c), Matthew Breetzke, Ryan Rickelton, Wiaan Mulder, Marco Jansen, Simon Harmer, Kagiso Rabada, Andrich Nortje.
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