Endogenous collagen cross-links influence cartilage biomechanical properties and resistance to degradation. Formalin fixation modifies collagen residues and forms new cross-links in a dose-dependent manner. We tested the hypothesis that magnetization transfer (MT) effects and T(2) depend on collagen cross-linking in cartilage. These parameters were measured in bovine nasal cartilage (BNC) prior to fixation, after 9 weeks of immersion in formalin solutions ranging in concentration from 0% to 10%, and after NaBH(3)CN reduction and washing. T(2) decreased by 59.4% +/- 1.1% upon fixation in 10% formalin, and was 32.2% +/- 5.2% shorter than initial values after washing. The apparent MT rate increased 25.9% +/- 3.7% and 52.8% +/- 7.1% over baseline under these conditions. Biochemical assays showed no significant differences in water, proteoglycan, natural cross-link, or collagen content between the 0% and 10% formalin-treated samples, while amino acid analysis demonstrated losses in (hydroxy)lysine and tyrosine, and new peaks consistent with methylene cross-links in fixed samples only. We conclude that formalin fixation of cartilage results in significant decreases in T(2) and increases in MT parameters that persist after removal of unreacted formaldehyde. The collagen cross-links thus created are associated with large changes in MT and T(2), indicating that interpretation of T(2) and MT values in terms of cartilage macromolecular content must be made with caution.