Neuro-2a neuroblastoma cells, when differentiated via a cAMP-dependent pathway by treatment with anti-GM3 monoclonal antibody, accumulated a high level of pp60c-src protein and pp60c-src kinase activity just before the onset of neurite formation. The specific kinase activity of the accumulated c-src protein was found to be comparable to that of normal cerebellar neurons, but was about 6- to 8-fold higher than that of normal astrocytes. These results, and migrations of peptide fragments in the SDS-polyacrylamide gels after V8 proteolysis, strongly indicate the accumulation of the neuron-specific isoform of the c-src protein (pp60c-srcN) in the GM3 antibody-treated Neuro-2a cells. Similar high levels of pp60c-src protein and pp60c-src kinase activity were observed in the Neuro-2a cells differentiated via a cAMP-dependent pathway by treatment with dibutyryl cAMP, but not in the same cell line when differentiated via a cAMP-independent pathway with 5-bromo-2'-deoxyuridine. These results demonstrate that the accumulation of high levels of the neuron-specific isoform of the pp60c-src protein (pp60c-srcN) in the Neuro-2a neuroblastoma cells depends on the specific signal transduction pathway involved during the differentiation of these cells.