Growing freshly dissociated chick-limb bud cells (stage 24) over agar for 48 hr permits differentiation into cartilage upon monolayer culture even when initial plating and subculture densities are well below confluency. Addition of 5-bromodeoxyuridine (BrdU) during the initial (48-hr) period over agar irreversibly inhibits chondrogenic differentiation, as characterized by morphology, metachromasia, and sulfate incorporation into acid mucopolysaccharide. Simultaneous, but not subsequent, addition of excess thymidine will prevent the effect of the analogue. Collagen synthesis is not depressed in BrdU-treated cells. Radioautographic studies demonstrate the specific localization of BrdU in the nucleus. Treatment of trichloroacetic acid-precipitable material containing tritiated bromodeoxyuridine with deoxyribonuclease solubilizes 90% of the radioactivity. The loss of the analogue from this precipitable material upon prolonged culture of limb-bud cells is more rapid than can be expected from cell division alone. 5-Bromodeoxyuridine may affect a fraction of DNA involved in stabilization of the differentiated cell phenotype.