C ardiac myocytes express the nitric oxide synthase isoform originally identified in constitutive nitric oxide synthase cells (NOS3), which mediates the attenuation by muscarinic cholinergic agonists of beta-adrenergic stimulation of L-type calcium current and contractility in these cells. However, calcium current and contractility in these cells. However, the reciprocal regulation of NOS3 activity in myocytes by agents that elevate cAMP has not been reported. In this study, we show that NOS3 and mRNA and protein levels in cardiac myocytes are reduced both in vitro after treatment with cAMP elevating drugs, and in vivo after 3 d of treatment with milrinone, a type III cAMP phosphodiesterase inhibitor. This effect on NOS3 activity by cAMP is cell type specific because treatment of cardiac microvascular endothelial cells in vitro or in vivo did not decrease NOS3 mRNA or protein in these cells. NOS3 downregulation in myocytes appeared to be at the level of transcription since there was no modification of NOS3 mRNA half-life by agents that increase intracellular cAMP. To determine the functional effects of NOS3 downregulation, we examined the contractile responsiveness of isolated electrically paced ventricular myocytes, isolated from animals that had been treated in vivo with milrinone, to the beta-adrenergic agonist isoproterenol and the muscarinic cholinergic agonist carbamylcholine. There was no difference in baseline contractile function in cells that had been pretreated with cAMP elevating agents compared to controls, but cells exposed to milrinone in vivo exhibited an accentuation in their contractile responsiveness to isoproterenol compared to controls and a loss of responsiveness to carbamylcholine. Downregulation of myocyte NOS3 by sustained elevation of cAMP may have important implications for the regulation of myocardial contractile state by the autonomic nervous system.