Thylakoid membranes transferred into a low water system composed of n-octane, the cationic surfactant cetyltrimethylammonium bromide (CTAB), and 1-hexanol as cosurfactant, displayed protein- and substrate-dependent ATPase activities for more than 60 min. This activity was enhanced 7-10-fold and 3-4-fold with 28%-vol. of methanol and 21 %-vol of tert-butanol present in the polar phase, respectively, in a fashion reminiscent of what occurs in aqueous media. Approximately 25% and 10% of control and methanol-enhanced ATPase activities found in buffer were detected in the low water system, respectively, and both activities showed a pronounced dependency on the amount of water present (between 12.5 and 15% of water (v/v )). H-Nuclear Magnetic Resonance (1 H-NMR) studies revealed that the bound/free water ratio (a) increased with decreasing concentration of water in the reverse micellar phase and (b) slightly increased in the presence of methanol. The results altogether suggest that the amount and physical state of water significantly contribute to determine the ATPase activity in the Low water system