Vitamin A and its congeners, collectively called retinoids, are known to have teratogenic potential and have induced craniofacial and limb malformations in numerous animal species. More importantly, retinoids are recognized as teratogenic to fetuses of pregnant women who have taken such preparations for dermatologic disorders. Information gathered from the study of animal models suggests that retinoids interfere with cartilage differentiation. If chondrogenesis in limb development is disturbed it may contribute to limb reductions and malformations. In vitro studies using various animal systems have shown that cartilage matrix macromolecules are altered to resemble those secreted by mesenchymal cells. The response of human chondrocytes to retinoids in vitro is not known. Culture of human chondrocytes in agarose maintains the cartilage phenotype and therefore serves as a model system to evaluate the influence of retinoids directly on human chondrogenesis. The studies presented in this paper were done to determine if the expression of specific matrix macromolecules of human chondrocytes in agarose culture is altered by retinol treatment. Immunocytochemistry demonstrated enhanced labeling of type I collagen while type II collagen labeling was reduced in cultures treated with retinol. In addition, morphometric analyses indicated a decrease in the size and number of chondrogenic clusters and that individual cells synthesized less alcian blue matrix when compared to parallel control cultures. The size of the proteoglycan monomers, glycosaminoglycan side chains as well as the disaccharide composition were not affected. However, there was a reduction in the quantity of proteoglycan monomers produced.