Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation.