Quail neural crest cells in vitro give rise to melanocytes and to adrenergic and cholinergic neurons. By supplementing the culture medium with heart cell conditioned medium (HCM), we stimulated cholinergic differentiation as indicated by a significant increase in the specific activity of the enzyme choline acetyltransferase. Melanin and catecholamine synthesis were concomitantly blocked as indicated by a lack of melanin granules and catecholamine-specific histofluorescence. Inhibition of pigmentation was fully reversible for at least 8 days, whereas inhibition of adrenergic differentiation became irreversible when cultures were exposed to HCM for more than 2 days. HCM also promoted cell attachment and spreading on the collagen substratum. These results suggest that HCM affected differentiation of quail neural crest cell progeny.