Human white blood cells produced interferon (IFN) in response to Staphylococcus enterotoxin B (SEB) in vitro. The majority of this IFN was classified as IFN-gamma by virtue of its acid instability, non-neutralization by antisera recognizing alpha or beta IFNs and species-specificity for human cells. The IFN activity appeared after 3-6 hr and reached maximum levels between day 2 and 3. The SEB-induced IFN was partially purified to about 10(5.5) units/mg protein by chromatography on controlled pore glass beads (CPG) and Sephadex G-100 columns. While the CPG column purification step increased the specific activity 50 to 100-fold, two peaks of IFN activity were eluted from the Sephadex G-100 column. The first activity peak containing the majority of the loaded IFN appeared to be active only on human cells and was not neutralized by anti-HuIFN-alpha and had an apparent molecular weight of about 44,000 daltons, the second peak (about 5% of the loaded IFN) showed antiviral activity on both human and bovine cells, was at least partially neutralizing by anti-HuIFN-alpha and had an estimated molecular weight of 21,000 daltons. The specific activity in the first IFN peak was about 30-fold increased by the gel filtration chromatography with a recovery of approximately 60-80%.