In rat pinealocytes, norepinephrine (NE) increases cAMP and cGMP accumulation through a synergistic dual receptor mechanism involving alpha 1- and beta-adrenergic receptors. The available evidence indicates that both increases in intracellular Ca2+ ([Ca2+]i, and activation of protein kinase C are involved in the alpha 1-adrenergic potentiation of beta-adrenergically stimulated cAMP and cGMP responses. In this study, the role of Ca2+ was further investigated using three agents with known anti-calmodulin activities: trifluoperazine, pimozide and W7. It was found that none of these inhibitors had any effects on the isoproterenol (ISO)-stimulated cAMP and cGMP responses. By contrast, all three Ca2+/calmodulin inhibitors reduced the NE-stimulated cAMP and cGMP responses. These results suggested that an alpha 1-adrenergic mediated mechanism was likely sensitive to the inhibitory action of the Ca2+/calmodulin inhibitors. To determine the possible site of action of the inhibitors, an activator of protein kinase C, 4 beta-phorbol 12-myristate 13-acetate (PMA), was used to potentiate the ISO-stimulated cAMP response. The PMA potentiation of ISO-stimulated cAMP response was only inhibited by a high concentration of pimozide, and not by trifluoperazine or W7, suggesting that pimozide may have other actions distinct from those of trifluoperazine or W7. Full potentiation of the cGMP response can be achieved by adding both PMA and a depolarizing concentration of K+. All three calmodulin antagonists were effective in inhibiting the PMA and K+ potentiation of cGMP responses in ISO-stimulated cells. These three compounds were also effective in inhibiting the potentiating effects of [Ca2+]i elevating agents on the beta-adrenergically stimulated cAMP and cGMP responses. The three inhibitors, at the concentrations used in the present study, were found to have no effect on the protein kinase C activity determined in vitro. These results indicated that apart from the protein kinase C pathway, the Ca2+/calmodulin pathway of signal transduction may be of importance in the regulation of pinealocyte cGMP and, to a lesser degree, cAMP responses.