We studied the in vitro immunomodulatory effect of sulfasalazine on purified human B cells, using a reversed plaque forming cell (PFC) assay and a proliferation assay. Sulfasalazine inhibited the PFC response of B cells in a dose-dependent manner. Sulfasalazine was added to PFC culture systems at several time points after the cultures were started. A marked reduction in B cell response was seen in the early phases (0-48 hours). Staphylococcus Aureus Cowan I (SAC) induced maximal B cell proliferation at day 3. Sulfasalazine at 5 micrograms/ml depressed that maximal proliferation on day 3. This indicates that sulfasalazine inhibited an early-phase event in the proliferation and differentiation of B cells. Sulfapyridine also inhibited the PFC response, but 5-aminosalicylic acid and N-acetyl sulfapyridine had no significant effect. These findings are significant since sulfapyridine is an active moiety of sulfasalazine, which is responsible for the second line of defense in the treatment of rheumatoid arthritis (RA). Adding T cells or macrophages to the PFC culture system had no significant effect. Furthermore, medium containing indomethacin was used to study the effect of prostaglandin released by residual macrophages. The results indicate that sulfasalazine inhibited the PFC response without affecting T cells, macrophages or prostaglandin. Sulfasalazine apparently has a direct immunosuppressive effect on B cells.