Guanosine 3':5'-cyclic monophosphate (cGMP) increased 7-fold in rat pineal glands incubated in the presence of l-norepinephrine. This response consisted of two components-one was stereospecific and inhibited by alpha-adrenergic antagonists while the other was not stereospecific and not readily inhibited by antagonists. Although l-isoproterenol was more potent than l-norepinephrine it had less intrinsic activity and its action was not stereospecifc. The increase in cGMP caused by these catecholamines, unlike that of adenosine 3':5'-cyclic monophosphate (cAMP), was dependent upon extracellular calcium. Ouabain and high levels of potassium produced a marked, calcium-dependent increase in pineal cGMP, without affecting cAMP. There was no effect of cholinergic agonists on cGMP. Surgical denervation markedly reduced the cGMP response to stimulation by l-norepinephrine, potassium, or ouabain. This was in contrast to the enhanced response of cAMP in denervated glands. The nonspecific increase in cGMP caused by l-isoproterenol, however, was not affected by denervation. These data demonstrate the existence of a calcium-dependent presynaptic mechanism for the generation of cGMP which may be mediated by an alpha-adrenergic-like receptor. In addition, the mechanisms regulating pineal cGMP appear to be physiologically distinct from those regulating cAMP.