Calciotropic hormones as well as biochemical parameters of bone formation and resorption show circadian rhythms. In a previous study in the rat, we observed a circadian rhythm in serum bone-resorbing activity (SBRA). In the present study, we investigated whether there was a circadian rhythm of SBRA in human serum. For this purpose, we studied 10 healthy premenopausal women and 5 healthy men. Blood was collected every 2 h, and urine samples were collected during 4-h periods for 24 h. For the determination of SBRA, media were prepared by reconstituting serum samples with Dulbecco's Modified Eagle's Medium at a ratio of 20% serum and 80% Dulbecco's Modified Eagle's Medium. Limb bones were dissected from 19-day old fetal rats prelabelled with 45Ca and were cultured for 72 h in the presence of the sera. Bone resorption was assessed from the 45Ca released into the culture medium and from that retained in the bone and was expressed as percentage 45Ca release. Serum calcium, phosphorus, PTH, cortisol, and urinary pyridinium cross-links were also determined. SBRA in human serum followed a circadian rhythm with a peak at about 0300 h and a nadir at 0700 h. There was no significant difference between the rhythm of SBRA of women and men. At concurrent time points, SBRA and serum PTH were positively correlated (r = 0.629; P < 0.01), and SBRA and serum cortisol were negatively correlated (r = 0.797; P < 0.01). To further investigate the possible contribution of these hormones to SBRA, either neutralizing anti-PTH antibody or RU-486 (mifepristone), a glucocorticoid receptor antagonist, was added to the serum samples of 6 subjects. Neutralizing the effect of PTH did not change the pattern of SBRA rhythm. The addition of RU-486 had a significant effect on the rhythm of SBRA, reducing the peak and nadir amplitudes. Thus we conclude that cortisol plays a major role in the rhythm of SBRA present in human serum; however, the influence of other factors cannot be excluded. Cortisol may be an important determinant of the circadian rhythm of bone resorption in vivo.