We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6-30 and 7-30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.