Elevated levels of bilirubin and other tetrapyrroles are common to a number of chronic hematologic and liver diseases that can result in growth failure. This report establishes a cellular model system for the study of these endogenous growth inhibitors. Primary chondrocyte cultures were prepared from embryonic chick cartilage; cells were incubated (0.3 ± 105 cells per plate) in tissue culture medium containing 10% fetal bovine serum (FBS) with or without added bilirubin, 0.01-0.10 mM. Bilirubin caused profound, dose-dependent inhibition of chondrocyte proliferation: after 7 days, control incubations contained 11.45 ± 2.0 ± 105 cells per plate versus 4.92 ± 0.55 ± 105 cells per plate for wells with added 0.01 mM bilirubin and 1.76 ± 0.30 ± 105 for cultures with added 0.05 mM bilirubin. In chondrocytes cultured for 3 days, the addition of 0.05 mM bilirubin was associated with inhibition of [3H]thymidine incorporation into DNA (47 ± 5% of control), [3H]uridine incorporation into RNA (11 ± 0.05% of control), and [14C]leucine incorporation into proteins (16 ± 1% of control). The inhibition of chondrocyte proliferation induced by a range of bilirubin concentrations (0.01-0.10 mM) was in no way attenuated by the addition of physiologic concentrations of albumin (4 g/dl). After 3 days in media containing bilirubin or heme (at equimolar concentrations), chondrocytes were subsequently incubated in FBS alone for an additional 3 days; only partial reversal of the bilirubin (or heme)-induced inhibition was then observed. Our data show that bilirubin (and heme) produces profound, dose-dependent, partially reversible inhibition of chondrocyte proliferation in culture without affecting cell viability, supporting the concept that these endogenous tetrapyrroles are potent growth inhibitors. We also conclude that chick chondrocytes in cell culture are a suitable model for delineating the mechanisms of tetrapyrrole tissue action.