Human and mouse lymphocytes produced IFN-α and IFN-α/β, respectively, when incubated with highly purified Staphylococcus aureus protein A (SPA). Maximum IFN induction occurred when lymphocytes were incubated with SPA at 250 to 500 μg/ml for 24 hr. The SPA induced-IFN were identified by neutralization of antiviral activity with specific antisera. A commercial preparation of SPA was found to induce both HulFN-α and HulFN-γ, with the latter comprising over 90% of the antiviral activity. The SPA preparation that induced IFN-γ was shown by SDS-polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay to be contaminated with 5% S. aureus enterotoxin A (SEA), a potent T lymphocyte mitogen and IFN-γ inducer. Preincubation of the commercial SPA with a specific antiserum to SEA blocked the induction of IFN-γ and had no effect on IFN-α production. SEA is a very potent IFN-γ inducer, effective at concentrations of 1 ng/ml or less. Thus, very minor contamination of SPA by SEA or other IFN-γ inducers could be responsible for previous reports that SPA induces IFN-γ in lymphocytes or that SPA behaves as a T cell mitogen.