Several studies in recent years have shown that the pyridinium crosslinks of collagen provide good urinary markers of collagen degradation, primarily reflecting bone resorption. Most studies, however, were based on time-consuming HPLC assays of the crosslinks. We now describe the development of an immunoassay (ELISA) based on a monoclonal antibody for free deoxypyridinoline (Dpd) and its use in healthy individuals and patients with bone-related disorders to measure the urinary excretion of Dpd as an improved assessment of bone resorption rate. The Dpd antibody exhibited less than 1% cross-reaction with free pyridinoline and was shown to react only with free Dpd in urine, having no significant interaction with peptide forms of the crosslinks. The intra- and interassay variations were less than 10 and 15%, respectively. A total of 402 urine samples from patients and healthy volunteers were analyzed by both the immunoassay and HPLC. The ELISA results were highly correlated with those for total Dpd measured by HPLC over the full range of sample groups (r = 0.95). In normal adults, the excretion of Dpd (mean + SD) was 4.7 + 1.6 nmol/mmol creatinine, with about fivefold higher excretion rates in children. For 31 osteoporotic patients, the ELISA Dpd values (median 6.7; range 3.0–13.5 nmol/mmol Cr) were significantly higher (p < 0.0001) than the corresponding values for age- and sex-matched controls (median 4.0; range 1.8–7.4). The difference between the groups was similar for total Dpd by HPLC (osteoporotic: mean 12.8, range 4.8–30.7; controls: 6.6, range 3.0–18.1; p < 0.0001). For other patient groups, comparisons of the Dpd excretion with healthy controls revealed similar differences for both the immunoassay and total Dpd analyses in primary HPT (n = 23: p < 0.0001), Paget's disease (n = 28, p < 0.0001), renal dysfunction (n = 26, no significant difference), and breast cancer (n = 17, p < 0.0001). We conclude that the immunoassay constitutes a simpler, more direct way of assessing bone resorption rates that provides similar information to the more cumbersome HPLC methods.