The ability of cytochalasin to influence the stimulation of murine B lymphocytes through surface immunoglobulin was assessed during short term cultures. Modest doses of anti-immunoglobulin alone did not stimulate proliferation of mouse spleen cells at 2 days. Cytochalasin B alone also had no effect. However, anti-immunoglobulin in combination with cytochalasin B stimulated substantial proliferation as judged by [3H]thymidine incorporation. Cytochalasins A, E, and D, and dihydrocytochalasin B were all effective in promoting B cell proliferation. Spleen cells from xid-defective (CBA/N X DBA/2)F1 male mice failed to proliferate in response to anti-immunoglobulin plus cytochalasin, suggesting that this treatment affects the same subset of B cells as anti-immunoglobulin plus B cell growth factor. Moreover, proliferation that was stimulated by anti-immunoglobulin plus cytochalasin B was not affected by T cell depletion. Cytochalasin may circumvent the need for, or replace, a second signal for proliferation.