The vitamin D endocrine system is crucial in calcium homeostasis in mammalian species. Central to this role 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) receptors have been detected in freshly isolated osteoblast-like bone cells and it has been shown that the active metabolite of vitamin D-3 1,25-(OH)2D3, increases bone resorption in vitro and in vivo. The requirement of 1,25-(OH)2D3 for the normal development of growth plate cartilage can be seen in vitamin D deficient rickets. However, there is still considerable controversy regarding the presence of 1,25-(OH)2D3 receptors in chondrocytes. In this paper, we report the presence of a 3.5-S 1,25-(OH)2D3-binding macromolecule in freshly isolated human costal but not articular chondrocytes. After subculture, both articular and costal chondrocytes have receptors. Saturation binding analysis revealed a single class of binding sites with an apparent Kd of 0.09 nM and approx. 2700 receptor molecules per cell for articular chondrocytes and a Kd of 0.1 nM and approx. 2000 receptor molecules per cell for costal chondrocytes. The presence of 1,25-(OH)2D3 receptors did not correlate with the switch from synthesis of cartilage-specific type II collagen to types I and III collagens. The acquisition of 1,25-(OH)2D3 receptors by articular chondrocytes may, therefore, be another phenotypic characteristic of cultured cells or may appear in vivo when chondrocytes are exposed to vascular or inflammatory cell products.