The subcellular organization and function of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores has been investigated in permeabilized hepatocytes using fluorescent probe techniques that monitor InsP3 action at the level of the Ca2+ storage organelles. Chlortetracycline fluorescence was used to follow alterations in luminal Ca2+, and InsP3-activated Mn2+ quench of compartmentalized fura-2 was used as a measure of the distribution and permeability of the InsP3-sensitive channels. Fluorescence imaging of single permeabilized hepatocytes attached to coverslips demonstrated that InsP3-sensitive Ca2+ stores are distributed throughout the cytoplasm, but are not present within the nuclear matrix. When hepatocytes were permeabilized in suspension, InsP3 activation of channel opening and Ca2+ release occurred in a quantal manner, such that the incremental magnitude of the response was determined by the dose of InsP3. Under these conditions dose-dependent steps of InsP3-induced Mn2+ entry into the stores occurred in the absence of changes in cytosolic or luminal Ca2+, providing evidence for a series of separate compartments with different sensitivities to InsP3. Electron microscopy studies revealed that the endoplasmic reticulum was extensively vesicularized when hepatocytes were permeabilized in suspension, whereas essentially normal endoplasmic reticulum structure was retained in cells attached to coverslips. In these attached cells the Ca2+ release and channel opening responses to InsP3 occurred in a nonquantal manner at the single cell level. Submaximal doses of InsP3 gave the same magnitude of response as a maximal InsP3 dose, although the rates of Ca2+ release and Mn2+ permeation through the InsP3-activated channels increased in a dose-dependent manner. Thus, in each cell the entire Ca2+ store was accessible for mobilization by all effective InsP3 concentrations. We conclude that the quantal release properties of the InsP3 receptor are not expressed in attached permeabilized liver cells because there is extensive luminal continuity within the InsP3-sensitive Ca2+ stores. This continuity appears to be disrupted when hepatocytes are permeabilized in suspension.