We have previously demonstrated that the nitric oxide (NO) donor S-nitroso-N-acetylcysteine (SNAC) reversibly decreases the activity of creatine kinase (CK) in an isolated rat heart preparation, markedly suppressing myocardial contractile responsiveness to an inotropic challenge. We wished to further examine the role of exogenous and endogenous sources of NO species on S-nitrosation of CK and subsequent enzyme activity in adult rat ventricular myocytes (ARVM). Two S-nitrosothiol groups were formed in the CK dimer after nitrosation of rabbit skeletal muscle CK in solution. CK inactivation due to S-nitrosation was time- and concentration-dependent in solution and in ARVM lysate for both NO donors S-nitroso-N-acetylpenicillamine (SNAP) and SNAC, and was rapidly reversible with the sulfhydryl dithiothreitol (DTT). Similarly, SNAC or SNAP dose-dependently decreased CK activity in intact ARVM, which was further attenuated by increasing the metabolic activity of the cells with electrical pacing for 1 h. Co-cultures of ARVM with interleukin 1 beta (IL-1 beta)- and interferon gamma (IFN gamma)-pretreated cardiac microvascular endothelial cells (CMEC) caused no detectable decline in myocyte CK activity. Increasing GSH levels attenuated the decline in myocyte CK activity with SNAC, while decreases in myocyte GSH levels enhanced the inhibitory effect of SNAC on intact myocyte CK activity. These data indicate that the degree of inhibition of cardiac myocyte CK by NO is dependent on the extent of myocyte metabolic activity and the intracellular GSH content.