Nine-day chick embryos were injected with a beta-xyloside and their sternal cartilage was examined 3 days and a week later. Sterna from 16-day embryos showed a reduction in size as compared to controls, with little or no change in the fraction of extracellular space, and a significant decrease in tensile strength. At the ultrastructural level, collagen fibrils in control sterna were dispersed evenly in the interstitial space, with few contacts between adjacent fibrils. In sterna from treated embryos, almost all collagen fibrils were aggregated into clumps and arrays throughout the interstitial space, with fibril-free areas in between. No abnormalities could be detected in the morphology of individual fibrils or in the ultrastructure of the chondrocytes. The changes in spatial distribution of collagen were fully evident 3 days after drug administration. The hydroxyproline/DNA ratio was the same in control and treated sterna, and no change was observed in the type of collagen. The uronic acid/DNA ratio was reduced by 14% 3 days after drug administration and by 40% after a week. The degree of sulfation of chondroitin sulfate was reduced from 80% in control sterna to 40% in treated sterna; almost all of this chondroitin sulfate was attached to peptide and the sedimentation pattern of the proteoglycan resembled that of normal cartilage proteoglycan. The function of chondroitin sulfate in embryonic cartilage is discussed in terms of our results and others. It is suggested that a major physiological role of the proteoglycan is to control the spatial distribution of collagen fibrils as they assemble to form a cross-linked gel.