Vitamin D appears to be required for mineralization of skeletal elements. There is also evidence that cartilage proteoglycans may be involved in the regulation of mineralization. Previous studies have shown an alteration in the structure of the proteoglycans of the epiphyseal growth cartilage as a result of the decrease in serum calcium related to deficiency of dietary vitamin D. Vitamin D deficiency also induces a thickening of the epiphyseal growth plate presumably because of the inhibition of maturation of the growth plate chondrocytes. In order to compare the effect on proteoglycan structure with that on growth plate morphology, the proteoglycans of healing epiphyseal cartilage were characterized. The results indicate that, consistent with previous data, in vitamin D-deficient hatching chicks, the proteoglycans of the growth cartilage, but not of the articular cartilage, are smaller in monomer size with slightly smaller chondroitin sulfate chains whose sulfation pattern is unaltered. Sternal cartilage proteoglycans are unaffected. During recovery from vitamin D deficiency, the proteoglycans isolated from the growth cartilage are still not completely normal one day after supplementation with vitamin D, but are indistinguishable from normal by four days. In addition, the results conflict with those of a previous study in which only growth cartilage of hatchling chicks, not sternal or articular cartilage, was reported to synthesize large proteoglycans. Instead, all of these cartilages in the normal chicken have been found in this study to produce large proteoglycans of a size typical for mammalian cartilage and embryonic chick cartilage.