Sections from the zone of calcification of ruthenium red-fixed normal avian epiphyseal growth plates were analyzed by various morphological (histochemical) and analytical techniques. Calcium and phosphorus were identified in the chondrocyte pericellular rim, the uncalcified extracellular (territorial) matrix, and in both the peripheral and central aspects of the calcified accumulations within extracellular matrix. Cartilage proteoglycan, as determined by ruthenium binding, positive staining with acidic phosphotungstic acid, and the X-ray spectroscopic detection of sulfur, was identified in the same four zones. Thus, it appears that proteoglycans, in some form, are indeed retained at sites of biological calcification. Additionally, these macromolecules, synthesized in chondrocytes, may be involved in extracellular calcium translocation.