The activity of enzymes exhibiting GTPase activity was determined in membrane preparations of hamster adipocytes. Two GTPases with apparent Km values of .apprx. 0.2 .mu.M GTP (low Km GTPase) and 180 .mu.M (high Km GTPase) were found. The effects of various agents that stimulate or inhibit adipocyte adenylate cyclase were investigated with these 2 forms of GTPase. None of the stimulatory or inhibitory agonists studied affected the activity of the high Km GTPase(s). Factors inhibiting adenylate cyclase, such as prostaglandin E1, nicotinic acid, 3-carboxy-5-methylpyrazole, and N6-phenylisopropyladenosine, stimulated the low Km GTPase by 50-100% without an apparent lag phase. The activity of the stimulated GTPase was half-maximal at .apprx. 0.2 .mu.M GTP. NaCl (up to 100 mM) had no effect on the basal activity of this enzyme but amplified the stimulation induced by adenylate cyclase inhibitory agents. There was a good correlation between inhibition of adenylate cyclase and stimulation of the low Km GTPase, both with regard to the concentration required for half-maximal effects on these 2 enzymes and with regard to the potency order of various prostaglandins studied. In contrast to factors inhibiting adenylate cyclase, the stimulatory hormones, isoproterenol and ACTH, had only small effects on the low Km GTPase activity; potassium fluoride was completely ineffective. An increased GTP hydrolysis by an activated GTPase apparently is an essential mechanism of hormone-induced adenylate cyclase inhibition.