Hormones have been demonstrated to activate phosphoinositide hydrolysis in plasma membranes in a manner dependent upon or potentiated by GTP. For thyrotropin-releasing hormone activation in GH3 cell membranes, stimulation persisted in membranes from pertussis toxin-treated cells. These observations indicate the presence of a membrane phospholipase C (PL C) and a novel GTP-binding protein (Gp); however, neither of these proteins has been characterized. In this paper, we report studies of GH3 membrane PL C utilizing [3H]phosphatidylinositol 4,5-bisphosphate liposome substrate. Guanosine 5'-O-(3-thiotriphosphate) (GTP[S]), but not other nucleotides, was found to stimulate PL C activity and required greater than 1 nM Ca2+. High concentrations of Ca2+ (10 microM) also activated the membrane PL C. Treatment of membranes with N-ethylmaleimide inhibited Ca2+-activated but not GTP[S]-activated PL C. Extraction of membranes with 1 M KCl solubilized the membrane PL C; however, the solubilized PL C was not GTP[S]-stimulated. N-ethylmaleimide-treated, KCl-extracted membranes were markedly deficient in GTP[S]-stimulated PL C activity; however, activity could be restored by incubation with the desalted extracted PL C. Reconstitution appeared to involve the recoupling of membrane-associated Gp with soluble 330- and 110-kDa forms of the PL C. Cytosolic PL Cs failed to substitute for the solubilized membrane PL C. These results indicate that the Gp-regulated PL C in GH3 cell membranes is an extrinsic membrane protein that can be extracted reversibly at high ionic strength. Moreover, the membrane PL C can be distinguished from cytosolic PL C isoenzymes.