The ability of 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (Smith et al., J Pharmacol Exp Ther 253: 688–697, 1992), to inhibit agonist-stimulated and store-operated Ca²⁺ inflow in single hepatocytes was investigated with the aim of testing whether the activation of phospholipase C is a necessary step in the process of agonist-stimulated Ca²⁺ inflow in this cell type. U73122 inhibited the release of Ca²⁺ from intracellular stores and plasma membrane Ca²⁺ inflow induced by vasopressin. An inactive analogue of U73122, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidone-dione (U73433), did not inhibit vasopressin-induced release of Ca²⁺ from intracellular stores, but did partially inhibit Ca²⁺ inflow. Neither U73122 nor ‘inactive’ analogue U73433 inhibited the release of Ca²⁺ from intracellular stores when this was initiated by the photolysis of ‘caged’ guanosine (5′-[γ-thio]triphosphate (GTPγS) introduced to the cytoplasmic space by microinjection. However, both compounds inhibited GTPγS-stimulated Ca²⁺ inflow. U73122 also inhibited the actions of glycerophosphoryl-myo-inositol-4,5-diphosphate (GPIP₂), a slowly-hydrolysed analogue of inositol 1,4,5-triphosphate (InsP₃) which is released by photolysis of ‘caged’ 1-(α-glycerophosphoryl)-myo-inositol-4,5-diphosphate, P⁴⁽⁵⁾-1-(2-nitrophenyl) ethyl ester, and thapsigargin in stimulating Ca²⁺ inflow. U73122 did not inhibit GPIP₂-stimulated release of Ca²⁺ from intracellular stores, but did partially inhibit the ability of thapsigargin to induce Ca²⁺ release. It is concluded that, while U73122 does inhibit phospholipase C_β in hepatocytes, complete inhibition of this enzyme in situ requires an intracellular concentration of U73122 higher than that achieved in the present experiments. Moreover, both U73122 and ‘inactive’ analogue U73433 have one or possibly two additional sites of action. These are likely to be the hepatocyte plasma membrane Ca²⁺ inflow channel protein (or a protein involved in the activation of this channel by the InsP₃-sensitive intracellular Ca²⁺ store), and a protein involved in thapsigargin action.