The putative phospholipase C inhibitor, U73122, transiently increases the cytosolic free Ca2+ concentration in rabbit pancreatic acinar cells by stimulating the release of Ca2+ from intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pflügers Arch. 427, 233-243]. In order to elucidate the exact mechanism of action of U73122 we studied its effects on both Ca(2+)-stimulated Mg(2+)-dependent ATPase activity and Ca(2+)-stimulated ATP-dependent Ca2+ uptake in rat liver microsomes. In addition, we studied its effects on Ca2+ release from steady-state loaded microsomes. The effects of U73122 were compared with those of thimerosal, described in the literature as inhibiting Ca(2+)-ATPases and sensitizing inositol 1,4,5-trisphosphate-operated Ca2+ release channels, and thapsigargin, a specific inhibitor of sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPases. Both U73122 (IC50 = 9 microM) and thimerosal (IC50 = 11 microM) dose-dependently inhibited Ca(2+)-stimulated Mg(2+)-dependent ATPase activity, without significantly affecting Mg(2+)-stimulated ATPase activity. Similarly, both U73122 (IC50 = 9 microM) and thimerosal (IC50 = 14 microM) dose-dependently inhibited ATP-dependent Ca2+ uptake. At concentrations beyond 20 microM, U73122 stimulated Ca2+ release from steady-state loaded microsomes at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin (1 microM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73122 concentrations cause an additional increase of passive Ca2+ leak. The data presented demonstrate that U73122 stimulates the release of actively stored Ca2+ primarily through inhibition of the internal Ca2+ pump.