Neural crest clusters were isolated after 18 or 42 h of growth in suspension and plated on a fibronectin substrate in the presence or absence of an overlay of a reconstituted basement membrane-like (RBM) matrix. After 1 week in vitro, cultures of 18-h clusters grown with the RBM gel overlay exhibited a 60-fold increase in the number of catecholamine-positive cells, while the number of melanocytes was decreased to one-quarter the control value. In contrast, clusters isolated after 42 h in suspension developed few adrenergic cells in the presence or absence of the RBM gel overlay. Melanocyte cell number was not altered in 42-h clusters grown in the presence of the RBM gel overlay. Total cell number after 1 week in vitro was similar under all conditions for both 18- and 42-h clusters. The distribution of tyrosine hydroxylase-immunoreactive cells among the experimental conditions was similar to that of catecholamine-positive cells. The presence of the neural tube was not necessary to generate the different developmental properties of clusters isolated at 18 h versus 42 h. These results show that the RBM gel can enhance adrenergic cell development and inhibit melanogenesis in young neural crest cluster cultures. Also, they demonstrate that the capacity of these neural crest cluster cell populations to differentiate and to respond to environmental cues changes as a function of time. These results are compatible either with a model in which the survival of adrenergic precursors within the clusters declines with time or one in which the adrenergic precursors switch their fate to another phenotype.