The potential effect of bilirubin on cartilage metabolism and growth in vitro was studied using embryonic chick and hypophysectomized rat cartilage bioassays, both very sensitive to cartilage growth factors and growth inhibitors. In chick cartilage, 0.25 mmol/L bilirubin caused significant but reversible inhibition of growth. Growth inhibition was due, at least in part, to bilirubin-induced inhibition of [35S]sulfate incorporation into proteoglycans by as little as 0.05 mmol/L bilirubin and to a lesser extent by inhibition of [14C]leucine incorporation into proteins and [3H]uridine incorporation into RNA. The addition of albumin or serum had no effect on bilirubin bioactivity. Thus, bilirubin may have a novel role in the pathophysiology of skeletal growth ratardation associated with some chronic diseases.