Rotation-mediated aggregating cell cultures of mechanically dissociated fetal (15-16 days gestation) rat brains between 25 and 35 days in vitro were examined for their ability to synthesize neurotransmitters and putative neurotransmitters from radioactively labeled precursors added to the culture medium. Cultures derived from whole brain synthesized [3H]acetylcholine from [3H]choline, [3H]gamma-aminobutyric acid from L-[3H]glutamic acid, [3H]dopamine from L-[3H]tyrosine, [3H]dopamine and [3H]norepinephrine from L-[3H]dihydroxyphenylalanine, and [3H]serotonin from L-[3H]tryptophan. Veratridine increased and tetrodotoxin decreased the rate of [3H]-dopamine synthesized by aggregates derived from midbrain plus hindbrain. In chase experiments in which aggregates were incubated for 4 h with radioactively labeled precursors and then for 4 h with non-radioactively labeled precursors, addition of veratridine (50 micronM) during the second 4 h incubation significantly decreased the amounts of radioactively labeled acetylcholine, L-glutamic acid, dopamine and serotonin recovered from aggregates. Tetrodotoxin (5 micronM) present during the chase significantly increased the amounts of [3H]acetylcholine and [3H]dopamine recovered from the aggregates. In addition, reserpine (4 micronM) markedly depleted [3H]dopamine from aggregates in these experiments. These results indicate that these cultured cells synthesized neurotransmitters and in addition suggest that some of these compounds are stored by and released from electrically active cells within the aggregates.