AbstractThe differentiation of neural crest cells into catecholaminergic neurons is dependent upon both intrinsic properties and signals from the embryonic microenvironment. In tissue culture, the development of catecholaminergic traits is dependent upon factors present in chick embryo extract (CEE). This dependency suggests that soluble growth factors affect catecholaminergic differentiationin vivo. We have studied the role of CEE‐derived factors and the potentially related influence of characterized growth factors on catecholaminergic phenotypic expression in avian neural crest cells. In this report, we show that CEE‐derived factors and transforming growth factor beta1(TGF‐β1) differentially influence catecholaminergic phenotypic expression as well as melanogenesis. TGF‐β1substituted for CEE‐derived factors and supported the in vitro differentiation of tyrosine hydroxylase (TH) and dopamine‐β‐hydroxylase (DBH) immunoreactivities, as well as catecholamine biosynthesis and storage. Differentiation of catecholaminergic cells was dependent on factors present in 10% CEE during the first 1–4 days in culture suggesting an initial critical period for exposure. One day of initial exposure to either CEE‐derived factors or TGF‐β1was sufficient to support the subsequent expression of catecholaminergic phenotypic characteristics. The time course of responsiveness to TGF‐β1was different than for CEE‐derived factors. Neural crest cells remain responsive to TGF‐β1for at least 5 days, which is past the critical period for CEE‐derived factors. Bioassay of CEE shows that endogenous levels of TGF‐β are less than or equal to 0.5 ng/ml. Immunoprecipitation of TGF‐β from CEE or blockade by neutralizing antibodies did not result in a loss of catecholaminergic differentiation by neural crest cells. Although CEE supports melanogenesis under all of the growth conditions tested, TGF‐β1was foun