Proto-oncogene Nck, an adapter molecule containing three SH3 and one SH2 domains, binds to cell surface receptors and mediates mitogenic effects in the cells. Overexpression of Nck caused cell transformation in vitro and tumor formation in the nude mice. The mechanism of this action by Nck, however, remained unclear. Rat adrenal pheochromocytoma cell line PC12 provides a useful system for studying growth factor-regulated cell proliferation and differentiation. Serum and epidermal growth factor (EGF) stimulate proliferation, whereas nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) cause growth arrest and sympathetic neurite outgrowth in these cells. To study the function of Nck, we generated stable clones of PC12 cells overexpressing the human Nck. We report here that the overexpressed Nck caused continued proliferation of PC12 cells even in the presence of NGF and blocked both the NGF- and bFGF-induced neurite outgrowth. Anti-sense but not sense oligonucleotides to the human Nck resumed the NGF-induced differentiation, indicating the specific inhibitory effect of Nck. Interestingly, Nck did not interfere with the kinetics of NGF- and EGF-stimulated protein tyrosine phosphorylation and the mitogen-activated protein kinase (MAPK) activation, suggesting that Nck inhibited the induced PC12 cell differentiation via a MAPK-independent mechanism. This study has provided a useful system for further understanding the function of Nck.