The mitogenicity, ability to induce immune interferon, and relationship between interferon synthesis and cell proliferative response were studied using human peripheral lymphocytes stimulated by staphylococcal enterotoxin A (SEA), phytohemagglutinin-P (PHA-P), and concanavalin A (ConA). Maximum cell proliferative responses ([3H]thymidine incorporation) and protein synthesis (14C-amino acid incorporation) occurred on days 3 and 4, respectively, after stimulation by each of the three mitogens. Maximal immune interferon levels were found 3 or 4 days after mitogen stimulation. SEA-treated cultures produced approximately three times more interferon than did cultures stimulated with PHA-P or ConA. Furthermore, SEA stimulated maximal cell proliferation over a much broader concentration range than did PHA-P and ConA (SEA, 10−5 to 102 μg/ml; PHA-P, 101 to 102 μg/ml; ConA, 101 to 101.5 μg/ml). Interferon was also produced at maximal or near maximal levels over a broad concentration range of SEA (10−2 to 102 μg/ml). Also, we found that inhibition of mitogen-induced DNA and protein synthesis to control levels by mitomycin C or cytosine arabinoside partially reduced interferon production. The DNA inhibitor studies indicate that immune interferon synthesis occurs maximally in association with at least some proliferative response and that submaximal levels of interferon production occur in mitogen-treated cultures in the absence of detectable proliferation. The ability of SEA to stimulate maximal DNA and immune interferon synthesis at concentrations of 3.5 × 10−13 M and 3.5 × 10−10 M, respectively, puts it in a potency range similar to that of hormones. Thus, SEA may play an important role in gut immunity and Staphylococcus aureus infections at concentrations well below those required for emetic effects.