In Tshwane, the metropolitan area that includes Pretoria, DA won 43.1 percent of the vote over the ANC's 41.2 per cent, according to final results in elections highlighting the declining popularity of the party that led the anti-apartheid struggle.
The loss of Pretoria comes on top of the ANC conceding defeat yesterday in Port Elizabeth, a key battleground of Wednesday's municipal election.
Defeat in Port Elizabeth by a margin of 46.7 to 40 percent was a humiliating blow for the ANC as the municipality is officially known as "Nelson Mandela Bay" in tribute to its past as a hotbed of anti-apartheid activism.
At the national level ANC remains the nation's top party. However, the party Mandela once headed has seen a slump in support after landing a national tally of 58.3 per cent of votes, a plunge of eight points from 2011.
DA leader Mmusi Maimane hailed the result as a landmark for his party.
"This is a tipping point for the people of South Africa," he said. "This represents for all of us the fact that we are a party not only of opposition but of government."
Turnout was about 58 per cent as voters chose mayors and other local representatives responsible for hot-button issues including water, sanitation and power supplies.
The setback to the ANC "happened quicker and harder than everyone thought! It's a shocker for everyone," said independent political analyst Ralph Mathekga of the worst poll result for the party since the end of white-minority rule 22 years ago.
Vice-president and party deputy leader Cyril Ramaphosa said the ANC would heed the electorate's message.
"Clearly our people are sending out messages all around, we are going to listen very, very carefully. We are a listening organisation, we are going to listen to our people," said Ramaphosa.
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