The response to SPA and Staphylococcus strain Cowan I (StaCw) of highly purified populations of peripheral blood and tonsil human lymphocytes was investigated. Purified T lymphocytes isolated from perpheral blood by E-rosetting were unable to respond in vitro to StaCw. Highly purified B-cell populations from tonsils did not show any proliferative response in the presence of soluble SPA. The addition to highly purified B-cell suspensions from human tonsils of increasing concentrations of autologous T lymphocytes did not induce any increase of thymidine uptake in the presence of StaCw. However, it was able to restore a marked proliferative response of the B-cell cultures to soluble SPA, even though mitomycin-treated T lymphocytes were added. The low response of highly purified peripheral blood T lymphocytes to soluble SPA could be potentiated by the addition of autologous mitomycin-treated B cells, whereas the unresponsiveness of purified T lymphocytes to StaCw was not affected. Mitogenic activity of SPA coupled to Sepharose beads was different from that of soluble SPA and paralleled that of StaCw. These data strongly suggest that insoluble SPA is a T-cell-independent B-cell mitogen in man, whereas soluble SPA, like PWM, exerts its activity on B cells only in the presence of T cells.