The present studies have established that there is an impaired response to epinephrine of the adenylate system in adipocyte preparations from obese hyperglycemic mice as compared to their thin littermates. In contrast, membrane preparations from both groups of animals were found to exhibit a similar response to fluoride ion. The response of adenylate cyclase to epinephrine was enhanced to a similar extent by increasing the ATP concentration in adipocyte plasma membranes from the two groups of animals. While GTP (0.1 muM) elicited an ATP-like response of similar magnitude in adenylate cyclase activity in both membrane preparations, it did not therefore abolish the impaired response to epinephrine of adenylate cyclase activity in membranes of obese mice. The response of adenylate cyclase activity to (--)-epinephrine in membrane preparations from obese mice progressively diminished with the age of these animals. In contrast, the concentration of (--)-epinephrine required for half-maximal stimulation of adenylate cyclase was similar and remained unchanged with the age for both membrane preparations. These data suggest that a perturbation may occur in the coupling step between the hormone receptor and the catalytic site of the adenylate cyclase system in obese mice. While a 15-day restrictive diet or a 72-h period of fasting was found to normalize the hyperinsulinemia of obese animals, neither affected the response of adenylate cyclase to epinephrine in preparations of adipocyte membranes from these mice. These results suggest that the observed defect in the response of plasma membrane adenylate cyclase activity to epinephrine in obese mice does not result from their hyperinsulinism.