Exposure of PC12 cells to nerve growth factor results in arrest of cell growth and induction of differentiation to sympathetic neuron-like cells, bearing neurites. In this study we identify a 48 kDa PC12 phosphoprotein as the neuron-specific protein kinase C substrate B-50 (Mr 48 kDa; IEP 4.5) on basis of comigration with purified B-50, immunoreactivity and phosphopeptide mapping. B-50 is present in both undifferentiated and differentiated PC12 cells. Exposure of PC12 cells to nerve growth factor for two days results in a 2.5-fold increase in the amount of B-50 as measured by RIA. Indirect immunofluorescence microscopy reveals that B-50 is mainly localized at the cell membrane and in growth cones. Our data are in line with the hypothesis that B-50 plays a role in neurite outgrowth and indicate that PC12 cells provide a suitable model to study this hypothesis.