The carbohydrate antigen recognized by monoclonal antibodies such as HNK-1 (first characterized as recognizing human natural killer cells) and NC-1 (raised against quail neural-crest-derived cells) is found on a number of molecules associated with cell differentiation in vertebrates . Previous work has shown that subpopulations of cultured quail trunk neural crest cells can be separated by fluorescence-activated cell sorting (FACS) on the basis of expression of this carbohydrate antigen. When neural crest cells are separated after 2 days in culture, adrenergic cells develop preferentially within the HNK-1-reactive subpopulation . We wished to investigate whether the capacity for adrenergic differentiation remained associated with the HNK-1-positive cell population at later times in vitro, when the percentage of HNK-1-reactive cells has declined. The present study found that neural crest cells separated according to HNK-1-reactivity after 4 days in culture also showed preferential development of adrenergic cells in HNK-1-positive-enriched cultures, indicating that the HNK-1 epitope is persistently expressed in vitro on cells with adrenergic potential after 4 days of culture. To investigate the possible function of this epitope in development of the adrenergic phenotype, HNK-1 was added to unsorted neural crest cell cultures. The presence of antibody resulted in a decrease in the percentage of HNK-1-reactive cells during the initial 24 h after replating, but had no effect on the number of catecholamine-positive cells which developed after 7 days. We conclude that the epitope recognized by the HNK-1 antibody does not appear to function in the induction of the adrenergic phenotype. However, this antigenic determinant is useful as a predictive early marker which defines a subset of neural crest cells that includes those with the ability to undergo adrenergic differentiation.