Biosynthesis of acetylcholine and gamma-aminobutyric acid was investigated in primary cultures derived from embryonic rat cerebral cortex. The cortical cultures contained a mixture of neurons and non-neuronal cells. Cortical neurons were viable for at least 2 months in vitro. The cultures accumulated [3H]choline from the medium and synthesized acetylcholine. Choline uptake was significantly reduced by hemicholinium-3 and acetylcholine synthesis by intact cultures was partially inhibited by naphthylvinylpyridine. In lysed cultures, greater than 90% of acetylcholine synthesizing activity was inhibited by naphthylvinylpyridine. Acetylcholine synthesis and storage by intact cells increased as the concentration of choline increased and approached saturation near 50 microM choline. Also, the synthesis and storage of gamma-aminobutyric acid from [3H]glutamate increased as the concentration of glutamic acid increased and began to saturate near 25 microM glutamate. The rates of synthesis of acetylcholine from 5 microM choline and of gamma-aminobutyric acid from 50 microM glutamate were linear for at least 30 min. The synthesis and accumulation of acetylcholine and gamma-aminobutyric acid from their respective precursors showed a similar dependence on culture age; they increased constantly during the first 3 weeks in culture, thereafter they declined. Mixed cultures of cortical cells and either skeletal muscle or various other non-neuronal cells exhibited a 40-100% enhancement in both acetylcholine and gamma-aminobutyric acid synthesis. Autoradiographic methods showed that a subpopulation of neurons in the cortical cultures accumulated gamma-aminobutyric acid from the medium.