Epidermal growth factor (EGF) induced short neurites in two different strains of PC12 cells. The length of the EGF-induced neurites was markedly increased in the presence of the protein kinase inhibitor K252a, which is known to inhibit differentiation induced by nerve growth factor (NGF). EGF-induced differentiation of PC12 required RNA synthesis and activity of the ras proto-oncogene product. EGF increased the levels of three neurofilament proteins and the mRNA level of two late response genes (SCG10 and 63) known to be induced by NGF. Together, EGF and K252a caused a greater increase in these mRNAs than did either agent alone. K252a did not alter the extent of EGF-induced autophosphorylation of the EGF receptor, but it did decrease the extent of receptor phosphorylation in the absence of added EGF. Thus, the ability of the EGF receptor to trigger neuronal differentiation may depend on the state of its phosphorylation at serine and/or threonine residues. Two other strains of PC12 did not extend neurites when exposed to EGF, even when K252a was also present. Thus, the differentiating effect of EGF on PC12 is PC12 strain-specific.