The acute effects of GH have a characteristic lag period of 10-30 min before a short lasting stimulation is seen, suggesting that some metabolic events precede the observable stimulation. There is evidence that cyclic nucleotides are involved in the mechanism of action of GH, which this work attempts to evaluate further. The effect of GH (rat, bovine, and human) on adenylate cyclase activity was measured in diaphragms and hearts of young normal and hypophysectomized rats. Measurements were made both after short term in vivo treatment and after addition directly to isolated muscle membranes. The muscles were homogenized and centrifuges, and the adenylate cyclase activity in the pellet was assayed. The injection of 4.5-450 pmol GH, iv, induced, within 3 min, inhibition of basal as well as epinephrine-stimulated adenylate cyclase activities, in both the absence and presence of a GTP analog, in the tissues studied. When added directly to the muscle membranes in the adenylate cyclase assay, 0.45-45 nM GH caused a dose-dependent inhibition of the adenylate cyclase activity, but only in the presence of a GTP analog. These findings clearly demonstrate that a physiological dose of GH has a rapid and direct inhibitory effect on muscle membrane adenylate cyclase, well within the lag period of the anabolic effect of the hormone.