The ability of cytochalasin to alter phosphoinositide metabolism was evaluated in naive, murine splenic B lymphocytes. The generation of total inositol phosphates was stimulated by treatment with cytochalasin D, as was the generation of inositol trisphosphate. The cytochalasin-induced increase in inositol phosphates appeared to depend on interaction with actin, and it was inhibited by phorbol esters. Inositol phosphate production was not stimulated in splenic T lymphocytes. The results suggest that the previously observed increase in intracellular calcium produced by cytochalasin is part of a more generalized signaling event that includes phosphoinositide turnover, and, further, raise the possibility of a functional association between actin and phospholipase C.