Previous studies have shown that in vitro calcification of chick epiphyseal cartilage matrix vesicles is proteolipid-dependent. The purpose of this research is to examine the role of proteolipid in cartilage calcification in vivo by comparing the proteolipid concentration of normal and vitamin D-deficient chick epiphyseal cartilage, the relationship of proteolipid to other tissue lipids, and its ability to support in vitro apatite formation. Proteolipid was isolated from the upper growth centers (reserve cell zone, upper proliferative zone) and lower growth centers (lower proliferative, hypertrophic, and calcified cartilage zones) of long-bone epiphyses from 3-week-old normal and rachitic male white leghorn chicks by Sephadex LH-20 chromatography of the crude phospholipid component of the total lipid extract. In both normal and rachitic tissue the proteolipid/dry weight and proteolipid/total lipid ratios were greater in the lower growth center than in the upper zones. No statistically significant change in the proteolipid/total lipid ratio in rachitic tissues relative to comparable cell zones in normal cartilages was observed. However, there was an increase in the nonproteolipid phospholipid content of rachitic tissues, altering the relative proteolipid/phospholipid composition. Whereas proteolipids from normal tissue supported in vitro calcification, proteolipids from rachitic tissues did not, indicating a direct effect of vitamin D on proteolipid structure. These data support the hypothesis that failure of rachitic cartilage to calcify in vivo may be due in part to alterations in phospholipid and proteolipid metabolism.