Nitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KC1. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KC1 is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2mM N-nitro-L-arginine methyl ester (L-NIV1AE), and 10mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KC1 under the depolarization conditions with 45mM KCI. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO.