The anionic detergent sodium polyanethole sulfonate was found to activate mouse B lymphocytes to increased cell division and formation of plaque-forming cells, possibly by an indirect mechanism. An increased cell division was also obtained in guinea pig and in rabbit lymphooid cell cultures. A strong similarity was found when comparing the dextran sulfate- and the sodium polyanethole sulfonate-sensitive cell population. Thus, the responding mouse cells resided in spleen and lymph node and also in fetal liver and bone marrow. The sensitive lymphocytes were dependent on the presence of macrophages or macrophage-replacing factors, since depletion of phagocytic cells resulted in loss of response. Addition of polymyxin B inhibited the stimulation, possibly through a direct interaction with the polyanion. In potency sodium polyanethole sulfonate was found to surpass dextran sulfate, making this benzene sulfonate a useful tool when studying lymphocyte responses.