The rapid release of Ca2+ from intracellular stores stimulated with inositol 1,4,5-trisphosphate (InsP3) has required superfusion or stopped-flow techniques to resolve the kinetics of Ca2+ mobilization and made it difficult to determine whether the InsP3 receptor desensitizes during prolonged stimulation. Here we have overloaded the intracellular Ca2+ stores of permeabilized rat hepatocytes by incubating them with ATP and 45Ca2+ in the presence of pyrophosphate, which precipitates Ca2+ within the lumen of the stores. Subsequent ATP removal initiated slow 45Ca2+ efflux that followed zero-order kinetics, allowing us to examine the effects of InsP3 over a prolonged time course. InsP3 produced a concentration-dependent increase in the 45Ca2+ efflux rate that was sustained for several min. The rate rapidly returned to the unstimulated level after the addition of decavanadate, a competitive antagonist of InsP3 at its receptor. Prior incubation with a submaximal concentration of InsP3 (1 microM) did not affect the subsequent enhanced rate of 45Ca2+ efflux stimulated by a higher, but still submaximal, concentration of InsP3 (3 microM). We conclude that prolonged exposure to InsP3 does not desensitize the InsP3 receptor and that intrinsic receptor desensitization cannot provide an explanation for the quantal responses to InsP3 observed in several cell types.