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During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995)Nature375, 68–73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21WAF1promoter and inducing expression of p21WAF1cyclin-dependent kinase inhibitor. NO activates the p21WAF1promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21WAF1promoter, expression of neuronal markers, and neurite extension. To determine whether p21WAF1is required for neurite extension, we prepared a PC12 line with an inducible p21WAF1expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21WAF1with isopropyl-1-thio-β-d-galactopyranoside restored this response. Levels of p21WAF1induced by isopropyl-1-thio-β-d-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21WAF1to block cell proliferation; and is required for neuronal differentiation by PC12 cells.