Primary cell cultures derived from embryonic rat brain were characterized by immunocytochemical methods using established cell markers and monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein, synapsin I. The cultures contained predominantly neurons, few astroglial cells and no oligodendroglial cells, based on immunocytochemical studies of the distribution of neuron-specific enolase, glial fibrillary acidic protein, myelin basic protein and galactocerebroside. Subsequently, the immunocytochemical localization of synapsin I, the cyclic GMP-dependent protein kinase and the various subunits of cyclic AMP-dependent protein kinase was determined. Synapsin I, a substrate for both the cyclic AMP- and Ca2+/calmodulin-dependent protein kinases, appeared particularly useful as a specific neuronal marker in primary cultures. Both immunocytochemical and immunoblotting techniques readily detected synapsin I in neuron-rich embryonic brain cultures, but indicated that synapsin I was absent from glia-rich primary cultures of newborn rat brain cells which lacked neurons. The intracellular localization of synapsin I in neurons changed markedly during the time of cell culture. In the first 10 days of cell culture, synapsin I appeared to be confined to neuronal cell bodies, whereas later it shifted to a patchy distribution in neuronal processes, perhaps indicating the transport of synapsin I in synaptic vesicles from the compartment of protein synthesis to its final synaptic location. Within neuron-rich embryonic cultures, the regulatory subunit (R-II) and the catalytic subunit (C) of cyclic AMP-dependent protein kinase appeared to be highly concentrated in neurons examined immunocytochemically. However, biochemical experiments demonstrated that R-II and C were also present in non-neuronal cell types of brain cell primary cultures. Cyclic GMP-dependent protein kinase, a marker protein for cerebellar Purkinje cells and for smooth muscle cells, was not detected immunocytochemically in neuron-rich cultures of embryonic brain cells, suggesting that Purkinje cells and smooth muscle cells were either absent from or not sufficiently developed in these cultures.