Metabolism of inositol 1,4,5-trisphosphate (IP3) and IP3-induced release of Ca2+ was analyzed in permeabilized human platelets. Both rapid Ca2+ release and hydrolysis of IP3 to inositol 1,4-bisphosphate (IP2) was observed after addition of IP3 to permeabilized, Ca(2+)-loaded platelets. In the absence of ATP or in the presence of inhibitors of the Ca(2+)-ATPase of the endoplasmic reticulum, no release of Ca2+ and little hydrolysis of IP3 occurs, indicating a coupling between the Ca2+ gradient across the membrane of the IP3-sensitive Ca2+ store and conversion of IP3 to IP2. In addition, the rapid recovery of the sensitivity of the IP3-sensitive Ca2+ store after successive additions of IP3 (increment detection) appears to be associated with hydrolysis of IP3.