Most voltage-gated Na+ channels inactivate almost completely at depolarized membrane potentials, but in some cells a residual Na+ current is seen that is resistant to inactivation. This persistent Na+ current can have a profound impact on the electrical behavior of excitable cells, and the regulation of this property could have important biological consequences. However, the biological signaling mechanisms that regulate the persistence of Na+ channels are not well understood. This study showed that in nerve terminals and ventricular myocytes nitric oxide (NO) reduced the inactivation of Na+ current. This effect was independent of cGMP, was blocked by N-ethylmaleimide, and could be elicited in cell-free outside-out patches. Thus, a reactive nitrogen species acts directly on the channel or closely associated protein. Persistent Na+ current could also be induced by endogenous NO generated enzymatically by NO synthase (NOS). Application of ionomycin to raise the intracellular Ca2+ concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with an NO-sensitive fluorescent dye. Persistent Na+ current was enhanced by the same treatment, and NOS inhibitors abolished both the elevation of NO and the induction of persistent Na+ current. These experiments show that NO is a potential endogenous regulator of persistent Na+ current under physiological and pathophysiological conditions.