The site, which is titled Africa Intelligence Leaks and contains no contact information, accused Public Protector Thuli Madonsela of being a CIA spy. She has previously denied the allegations.
Julius Malema, leader of an opposition party that says it wants to redistribute resources to the poor, was also accused of working for American intelligence.
The US Embassy in South Africa said it had no comment. The CIA also declined to comment.
The State Security Agency, South Africa's intelligence service, said it viewed the matter seriously.
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Madonsela released a report last year that said President Jacob Zuma had inappropriately benefited from state funding and should pay back some of the more than USD 20 million spent on alleged security upgrades at his private home.
Madonsela received praise in some quarters, but her detractors accused her of posturing in the media and pandering to the political opposition.
Madonsela's office demanded that the deputy minister produce evidence of his claims. The South African government distanced itself from Maphatsoe's comments and he apologized.
Malema is a former head of the ANC's youth league who was kicked out of the party and formed the Economic Freedom Fighters, which entered parliament in elections last year.
Mbuyiseni Ndlozi, spokesman for Malema's party, criticized the government decision to investigate the spying allegations. "They should focus on matters of state security rather than looking for grounds to investigate opposition leaders," Ndlozi said.