Specific hydrolysis of GTP catalyzed by membranes prepared from A6 epithelial cells grown on porous supports was measured. Aldosterone treatment of the cells for 4 h increased Na+ transport and stimulated GTP hydrolysis by apical membranes in vitro more than twofold over basal levels. This stimulation was attributed to an increase in maximum velocity with little change in Michaelis-Menten constant values. Na+ transport rate and GTP hydrolysis were linearly correlated after aldosterone. This relationship was maintained when aldosterone's response was blunted by various inhibitors. Spironolactone decreased both the hormone-stimulated guanosinetriphosphatase (GTPase) and the Na+ transport rate. Pertussis toxin, which exerted minimal effects on basal rates, reduced the increase of Na+ current normally observed after aldosterone and the hormone stimulation of GTPase activity. The expression of classical Gi/Go-type G proteins was not increased after hormone treatment. When A6 cells were grown on nonporous plastic dishes, aldosterone neither stimulated GTPase activity nor increased amiloride-blockable 22Na+ fluxes. We propose that activation of one or more G proteins in the apical membrane of A6 cells is directly involved in the natriferic action of aldosterone.