THE effect of the GTP-analogue guanylyl 5′-imidodiphosphate (Gpp[NH]p) on [3H]forskolin binding was studied in rat brain using autoradiography. In the striatum, 100 μM Gpp[NH]p produced a 40% increase in binding, whereas a decrease of about 30% was observed with low Gpp[NH]p concentrations (0.1–1 μM). In the molecular layer of the cerebellum all concentrations of Gpp[NH]p decreased [3H]forskolin binding. The decrease in binding disappeared in both striatum and the molecular layer of cerebellum in sections pretreated with 100 m μM N-ethylmaleimide (NEM) for 10 min. NEM pretreatment did not significantly affect the stimulation of [3H]forskolin binding by micromolar concentrations of Gpp[NH]p in the striatum, but reversed the decrease observed in the molecular layer of the cerebellum, to an increase. Based on these data we suggest that the effects of the GTP-analogue Gpp[NH]p on [3H]forskolin binding may involve both Gs and Gi, where a stimulation produces an increase and decrease in binding respectively. The regional effects of Gpp[NH]p may reflect differences in the responsiveness of adenylyl cyclase to Gs and Gi-mediated effects.