Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4-5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated 3H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release 3H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca2+ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10-11 days in vitro. The fully expressed release capacity (20-30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.