Vitamin C is essential for the hydroxylation of lysine in collagen. The resulting hydroxylysine residues, together with the precursor lysine residues, are then incorporated into the stable collagen cross-links: pyridinoline and deoxypyridinoline. A reduction in lysine hydroxylation, resulting from an inadequate supply of vitamin C, might decrease the ratio of the pyridinoline crosslinks to the deoxypyridinoline crosslinks. This hypothesis was tested in a guinea pig model, using a purified diet providing vitamin C in the following amounts: (a) 0.5 mg/day; (b) 1 mg/day; (c) 5 mg/day; (d) 0.25% w/v in the diet (providing 49 mg/day); (e) 1.0% w/v (providing 205 mg/day); or (f) 5.0% w/v (providing 1128 mg/day). Weanling male guinea pigs each received one of these diets for 46 days, after which their tissue vitamin C concentrations, and the concentrations of hydroxyproline, pyridinoline, and deoxypyridinoline in bone, and of creatinine, pyridinoline, and deoxypyridinoline in urine, were measured. The ratio of pyridinoline to deoxypyridinoline in bone collagen and in urinary collagen fragments decreased significantly as vitamin C depletion became more severe. Animals receiving the higher intakes of vitamin C grew more rapidly than did those receiving the lower intakes, but the relationship between vitamin C intake and the collagen crosslinks ratio remained highly significant, even after adjusting for intergroup differences in growth rates. Thus, the ratio between the two collagen crosslinks could provide a specific and sensitive functional index of vitamin C in animals and man. This is an important goal, in view of the need to be able to define optimum intakes of vitamin C.