The activity of epinephrine-sensitive adenylate cyclase of human fat cell ghosts is markedly enhanced by the GTP analog 5'-guanylyl-imidodiphosphate (GMP-P(NH)P), but a similar effect of GTP itself has not been heretofore demonstrable. In the present work, comparison of adenylate cyclase activity in the presence of epinephrine alone versus epinephrine plus GTP showed that at 37 degrees C GTP doubled activity (10-min incubation); at 30 degrees C less than half this effect was apparent. However, time course studies at both 30 and 37 degrees C showed that comparisons at a single point in time based on ratios of hormone-stimulated activity to basal or basal plus GTP were misleading, since basal activities were not linear with time and were inhibited by GTP. The inhibitory effect of GTP on basal activity was seen at both temperatures but at 37 degrees C decreased with time so that by 10 min the inhibition was no longer apparent. The time course data showed clearly that epinephrine alone did not stimulate adenylate cyclase activity; rather, the hormone merely prevented fall-off of initial rate of unstimulated (basal) enzyme activity. Only when GTP was added together with epinephrine was an unequivocal stimulation of enzyme activity observed. GTP effect was dose-dependent with half-maximal enhancement of epinephrine stimulation at 1.0 microM GTP. The GTP effect was not hormone-receptor mediated, since no shift was seen of the epinephrine dose-response curve toward higher sensitivity. GTP enhancement of epinephrine stimulation occurred over a wide range of ATP concentrations (0.01-3.0 mM) and affected the substrate Km only minimally. GTP-enhanced activity thus occurred through increased V max of the hormone-sensitive adenylate cyclase.