The roles of the heterotrimeric G-protein, G(i2), in regulating the actin cytoskeleton and the activation of store-operated Ca2+ channels in rat hepatocytes were investigated. Gα(i2) was principally associated with the plasma membrane and microsomes. Both F-actin and Gα(i2) were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant coprecipitated with Gα(i2) using either anti-Gα(i2) or antiactin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some Gα(i2) co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70-80% of Gα(i2) in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca2+ inflow. It is concluded that (i) a significant portion of hepatocyte Gα(i2) associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca2+ inflow.