Recombinant protein A(SpA) produced in Escherichia coli or Bacillus subtilis bacteria did not induce activation of human peripheral mononuclear cells, whereas SpA preparations obtained from naturally occurring Staphylococcus aureus bacteria as well as recombinant SpA from Staphylococcus xylosus were potent mitogens. Further purification of SpA from S. aureus showed that the mitogenic material was concentrated in the side fractions containing more basic molecules. Some staphylococcal enterotoxins are mitogenic for human cells and in order to test whether contaminating enterotoxins would be responsible for the mitogenic effect of SpA preparations, rabbit antibodies were produced against enterotoxin A and B. These antibodies inhibited activation of human cells induced by the enterotoxins used for immunization but did not affect the activation induced by SpA preparation. The addition of selected human sera to in vitro cultures resulted in an inhibition of the response induced by low doses of SpA. There was no clear relationship between these effects and the content of IgG antibodies against staphylococcal enterotoxins A, B, and Cl in the sera. Thus, we conclude that the mitogenicity of SpA preparations is caused by contaminating molecules, probably not enterotoxins A, B, or Cl.