Objective: Little is known about how the biochemical properties of collagen change during tissue regeneration following cartilage damage. In the current study, temporal changes in cartilage repair tissue biochemistry were assessed in a rabbit osteochondral defect. Design: Bilateral full-thickness 3-mm osteochondral trochlear groove defects were created in 54 adult male skeletally mature New Zealand white rabbits, and tissue repair was monitored over 16 weeks. Collagen content, cross-links, lysyl hydroxylation, gene expression, histological grading, and Fourier transform infrared analyses were performed at 2, 4, 6, 8, 12, and 16 weeks. Results: Defect fill occurred at ~4 weeks postinjury; however, histological grading showed that the repair tissue never became normal, primarily due to the presence of fibrocartilage. Gene expression levels of Col1a1 and Col IIaI were higher in the defect compared with adjacent regions. Collagen content in the repair tissue reached the level of normal cartilage at 6 weeks, but it took 12 weeks for the extent of lysine hydroxylation to return to normal. Divalent immature cross-links markedly increased in the early stages of repair. Though the levels gradually diminished thereafter, they never returned to the normal levels. The mature cross-link, pyridinoline, gradually increased with time and nearly reached normal levels by week 16. Infrared imaging data of protein content paralleled the biochemical data. However, collagen maturity, a parameter previously shown to reflect collagen cross-link ratios in bone, did not correlate with the biochemical determination of cross-links in the repair tissue. Conclusion: Collagen biochemical data could provide markers for clinical monitoring in a healing defect.