The stimulation of hepatocytes by alpha 1-adrenergic agonists and vasoactive peptides results in a mobilization of intracellular Ca2+ which is accompanied by breakdown of phosphatidylinositol 4,5-bisphosphate to release myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The possible involvement of Ins(1,4,5)P3 in intracellular Ca2+ mobilization was tested using a preparation of saponin-permeabilized hepatocytes. Added Ca2+ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca2+ concentration was lowered to a new steady state level. The subsequent addition of Ins(1,4,5)P3 caused a rapid Ca2+ release, which was complete within 5 s. Half-maximal and maximal Ca2+ release were obtained at concentrations of Ins(1,4,5)P3 of 0.1 and 0.5 microM, respectively. The maximal amount of Ca2+ mobilized was 450 pmol/mg of cell dry weight. Using experimental conditions designed to permit selective Ca2+ accumulation into mitochondrial or non-mitochondrial stores, it was determined that all of the Ca2+ released by Ins(1,4,5)P3 originated from non-mitochondrial, vesicular stores. After Ca2+ release was completed, reaccumulation occurred until the medium free Ca2+ concentration was restored to its original level. Experiments using 32P-labeled Ins(1,4,5)P3 indicated that Ca2+ reaccumulation was associated with dephosphorylation of this compound. From a consideration of the properties of the Ca2+ release induced by Ins(1,4,5)P3, with respect to its kinetics, dose response, specificity, and the amount of Ca2+ released, the data strongly suggest that this compound is a second messenger involved in the hormonal mobilization of Ca2+ from intracellular stores.