Stimulation of human platelet adenylate cyclase by the diterpene forskolin is associated with a decrease in the apparent substrate (MgATP) affinity of the enzyme. Addition of the stimulatory hormone prostaglandin E1 not only further increased the Vmax. of the forskolin-stimulated platelet adenylate cyclase but also caused a further increase in the Km value for MgATP, by up to 20-fold compared with basal conditions. On the other hand, the inhibitory hormone adrenaline decreased not only the Vmax. but also the Km value of the platelet adenylate cyclase stimulated by forskolin, with or without prostaglandin E1 present. The data indicate that forskolin sensitizes human platelet adenylate cyclase to modulation of substrate (MgATP) affinity by hormones, but there is no such effect in the absence of the diterpene.