The effects of several non-neuronal cell types on neurotransmitter synthesis in cultures of dissociated sympathetic neurons from the new-born rat were studied. Acetylcholine synthesis from radioactive choline was increased 100- to 1000-fold in the presence of non-neuronal cells from sympathetic ganglia. This increase was roughly dependent on the number of ganglionic non-neuronal cells present. The effect did not appear to be due to an increased plating efficiency of neurons, since the non-neuronal cells were capable of increasing acetylcholine synthesis after only 48-hr contact with neurons that had been previously grown without non-neuronal cells for 2 weeks. C6 rat glioma cells were also able to stimulate acetylcholine synthesis, but 3T3 mouse fibroblast cells had little or no effect. None of the non-neuronal cell types synthesized detectable acetylcholine in the absence of the neurons. The ganglionic non-neuronal cells had no significant effect on catecholamine synthesis (which occurs in the absence of non-neuronal cells).