Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.