Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60Jcsrc. The 60-kilodalton (kDa) form appeared similar to pp6Ocsrc from cultured rat fibroblasts or astrocytes.The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60csrc was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60Csrc was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60csl`population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60CSrC and the increase in specific protein kinase activity may be important for neuronal differentiation.Cellular genes homologous to retroviral oncogenes are present in the genomes of all vertebrates. These cellular proto-oncogenes have been highly conserved throughout evolution, suggesting that they are essential to the organism. Although their function remains largely unknown, evidence is accumulating that some may be important for normal cell differentiation or growth.The cellular gene c-src is homologous to the transforming gene of Rous sarcoma virus (59). It is highly conserved phylogenetically, being present in the genome of such widely divergent species as humans, Drosophila melanogaster (35,61,62), and the freshwater sponge Spongilla lacustris (3, 55). The c-src gene encodes a 60-kilodalton (kDa) membraneassociated phosphoprotein, pp6Oc-src, which is a proteintyrosine kinase (14-17, 24, 36, 38, 49, 52, 59). Evidence is emerging that pp60C-src is the product of a developmentally regulated gene that may participate in cell differentiation. High levels of pp60csrc are expressed in brain and other neural tissues of both chickens and humans during embryogenesis (22,42,45). Expression of pp60-src in the developing chick neural retina (64) and cerebellum (29) coincides with the onset of neuronal differentiation. Post mitotic neurons from the central nervous system of rat embryos express high levels of a structurally distinct, enzymatically activated form of pp60C-src (9). Similarly, embryonal carcinoma cells induced to differentiate into neuronlike cells have elevated levels of a slower-migrating form of pp60c-src (44). There are other nonproliferating cells, for example, platelets (31) and myeloid cells (2, 30), which contain increased pp6Oc-src kinase activity. The presence of high levels of pp6Oc-src protein-tyrosine kinase activity in * Corresponding author. these nondividing cells suggests that pp60csrc may be importan...