The modulatory role of endogenous cellular glycosphingolipids in bradykinin-stimulated myo-inositol 1,4,5-trisphosphate (InsP3) formation by MDCK cells was evaluated utilizing the glucosylceramide synthase inhibitor, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Bradykinin-stimulated InsP3 formation in intact cells and in isolated plasma membranes was significantly enhanced when cells were first depleted of their glucosphingolipids. The effect of glucosphingolipid depletion on phospholipase C activity was dependent on the duration of exposure to the inhibitor and the cellular level of glucosylceramide. Inclusion of glucosylceramide in the culture medium prevented the stimulatory effect of PDMP on InsP3 formation. It is concluded that membrane glucosphingolipids may regulate phospholipase C activity.