Polyclonal antibody was raised in rabbits against a synthetic hexapeptide R-K-V-D-V-R corresponding to a unique amino acid sequence of the neuron-specific c-src gene product pp60c-src(+). The antibody was purified by affinity chromatography. A single band with an apparent molecular mass of 60 kDa was recognized when the supernatant of homogenates of brain and spinal cord from chick embryos and chicks was probed with the affinity purified anti-hexapeptide antibody after SDS-polyacrylamide gel electrophoresis followed by Western blotting. Specificity of the antibody was further characterized by autophosphorylation assay of immunoprecipitate in comparison with the monoclonal antibody 327. Immunocytochemical studies by light microscopy revealed that pp60c-src(+) was localized in flake-like aggregates in neuronal cell bodies of the spinal cord in 7-15-day-incubated chick embryos and newly hatched chicks. Developing spinal ganglia and muscle cells were also immunoreactive at early developmental stages. By electron microscopy, the reaction product was observed mainly in two regions. One region was at polysomes and along the membranes of the rough endoplasmic reticulum. The other region was along the neuronal plasma membrane--at subsurface cisterns and at synapses. At synapses, the postsynaptic density, presynaptic membrane and synaptic vesicle membranes were immunostained. Immunoreactivity at synapses were more frequently observed at earlier stages than at later stages of development. These findings suggest that pp60c-src(+) is actively produced in developing neurons and has some important roles in synaptogenesis. In mature synapses, pp60c-src(+) may be involved in the interaction of synaptic vesicles with the presynaptic membrane.