We have previously shown that microfilament-disrupting agents inhibit steroid secretion by frog adrenocortical cells. To determine the role of microfilaments in the process of corticosteroid production, we studied the effects of cytochalasin-B and chaetoglobosin-C on polyphosphoinositide metabolism in myo-[3H]inositol-prelabeled frog interrenal (adrenal) slices. Immunocytochemical labeling of adrenocortical cells in primary culture with actin antiserum showed that cytochalasin-B (5 x 10(-5) M) induced a complete and reversible disruption of microfilaments, whereas chaetoglobosin-C, a cytochalasin analog that cannot interact with actin, did not modify the organization of the microfilament network. Cytochalasin-B caused a dramatic inhibition of corticosteroid release from perifused frog interrenal slices, whereas chaetoglobosin-C did not affect steroid secretion. Analysis of labeled inositol phosphates and phosphoinositides revealed that cytochalasin-B, but not chaetoglobosin-C, caused a significant increase in tritiated inositol content (+38%) and concurrently inhibited the formation of polyphosphoinositides (-48%). Cytochalasin-B reduced the production of phosphatidylinositol (-63%), phosphatidylinositol monophosphate (-46%), phosphatidylinositol bisphosphate (-46%), and lyso-phosphatidylinositol (-66%). Cytochalasin-B also blocked the stimulatory effect of angiotensin-II on the breakdown of phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate and the formation of lyso-phosphatidylinositol and inositol phosphates. The present results provide evidence of a role for microfilaments in polyphosphoinositide metabolism in adrenocortical cells. These data indicate that microfilaments are required for the incorporation of inositol into membrane phospholipids and are necessary for angiotensin-II-induced phospholipase activation.