To investigate whether bone resorption exhibits a diurnal variation in healthy premenopausal women, we measured urinary excretion of pyridinoline and deoxypyridinoline cross-links (U-Pyr/Cr and U-D-Pyr/Cr) every 3 h over a 24-h period in 12 healthy premenopausal women (mean +/- 1 SD age, 32 +/- 5 yr). To study diurnal variation, U-Pyr/Cr and U-D-Pyr/Cr are the best available markers of bone resorption for a diurnal variation study, as they are not influenced by diet. Plasma osteocalcin and serum alkaline phosphatase (markers of bone formation) were also measured. A marked diurnal rhythm was observed in U-Pyr/Cr and U-D-Pyr/Cr, with the peak between 0500-0800 h and the nadir between 1400-2300 h. The fluctuation was 2-fold over the 24-h period, with a mean fall of 25-35% between 0800-1100 h. This study demonstrates a marked diurnal variation in the new biochemical markers of bone resorption, U-Pyr/Cr and U-D-Pyr/Cr, in premenopausal women. Furthermore, this study emphasizes the importance of regulating the time of the urine sample taken for measuring urinary pyridinium cross-links for clinical investigations as well as in clinical practice.