Gs and adenylate cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with or without 1000-fold transmembrane Ca2+ gradient. Obtained results showed that Gs activities of both binding GTP gamma S and stimulating adenylate cyclase were the highest in proteoliposomes, with a transmembrane Ca2+ gradient similar to the physiological situation and the lowest while the transmembrane Ca2+ gradient was in the inverse direction. Such a difference could be diminished following the dissipation of the transmembrane Ca2+ gradient by A23187. Time-resolved fluorescence anisotropy of diphenylhexatriene (DPH) has been used to compare the physical state of phospholipids among those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for higher membrane fluidity, which may favor Gs function with higher GTP-binding activity and stimulation of adenylate cyclase.