Fragments of the alpha1 C-terminal telopeptide of type I collagen containing the sequence AHDGGR(1209-1214) (CTx) can be measured in urine as an index of bone resorption. We report here that these molecules undergo racemization and isomerization of Asp(1211) in vitro and in vivo, generating a mixture of four isomers: the native peptide form (alphaL), an isomerized form containing a beta-Asp bond (betaL), a racemized form containing a D-Asp residue (alphaD) and an isomerized/racemized form (betaD). To study these reactions at this specific site in collagen, we have employed four immunoassays, each specific for one of the isoforms, and developed HPLC methods for their separation. The kinetics of these reactions were studied in vitro under physiological conditions by incubation of synthetic AHDGGR hexapeptide or mineralized bone collagen. Reactions were found to be strongly shifted towards the beta-Asp forms and slightly in favour of the D-enantiomeric forms. CTx isomers were measured in human urine and in enzymic digests of bovine bone collagen. The results indicated that the extent of racemization and isomerization were correlated with the age and turnover of collagen. The ratios between the native and age-related forms of CTx were elevated in urine from patients with Paget's disease or osteoporosis as compared with that from healthy adults. The alphaL/alphaD CTx ratio had the highest discriminatory power (T-score=23.2; P<0.0001 and T-score=1. 5; P<0.0001 for Paget's disease and osteoporosis respectively). In conclusion, these findings indicate that an assessment of CTx ratios in urine may provide an estimate of bone turnover, aiding in the diagnosis of metabolic bone diseases.