Formation of calcium pyrophosphate dihydrate (CPPD) crystals in native collagen gels represent an in vitro model system for the study of pathological cartilage calcification. The conditions under which CPPD forms in collagen gels have been determined. At low Ca X pyrophosphate product, CPPD forms directly. At high Ca X pyrophosphate product, CPPD forms via the amorphous intermediate calcium magnesium pyrophosphate (CMPP). Chondroitin sulfate (CS) inhibits formation of CPPD by both pathways, but apparently by different mechanisms. Direct CPPD formation is inhibited with low potency by CS, apparently by binding of Ca2+ ions. Indirect formation of CPPD is inhibited with high potency by CS, apparently by stabilization of the CMPP intermediate. Comparison of the inhibition of direct CPPD formation by the two glycosaminoglycan species occurring in cartilage proteoglycan showed that CS is a more potent inhibitor than keratan sulfate (KS), in agreement with the greater Ca2+-binding affinity of CS. The increase in KS/CS ration which occurs in human hyaline cartilage with aging may facilitate deposition of CPPD crystals by decreasing the exclusion of pyrophosphate anions.