Human erythrocyte membranes were incubated in the presence of sodium fluoride or guanylylimidodiphosphate (GppNHp), a nonhydrolysable GTP analog. After centrifugation at 100000 g the activity of the adenylate cyclase-stimulating GTP-binding protein, Gs, was detected in the supernatant fraction. The release of the Gs activity from the membranes closely resembles Gs activation by GppNHp. The Gs activity release from the GppNHp-induced membranes is characterized by a lag period. The nucleotide concentration causing a half-maximal solubilization is about 9.10(-7) M. Approximately 50% of the Gs activity released from sodium fluoride-treated human erythrocyte membranes was associated with the cytoskeletal fraction extracted by a low ionic strength solution. The data obtained suggest that Gs exists in the membrane at lease in two compartmentalized states and is solubilized from both states during its activation.