Urinary excretion of bone labels can be used to monitor bone resorption. Here we investigate the effects of dosing frequency on label incorporation of various sites when bone turnover was perturbed by ovariectomy. We compared tritiated tetracycline ((3)H-TC) and (45)Ca in two studies. Nine-month-old rats were given single or multiple injections of (3)H-TC and (45)Ca and sacrificed after 7 or 14 days. Six-month-old OVX rats were given (3)H-TC and (41)Ca tracers 1 or 3 months following ovariectomy (OVX + 1 mo or OVX + 3 mo, when bone turnover was higher or lower, respectively) and sacrificed 1 week, 1 month, 3 months, or 6 months postdose. Twenty-four-hour urine pools over 2-4 consecutive days as well as the proximal tibia, femur midshaft, lumbar vertebrae (L1-L4), and remaining skeleton were analyzed for (3)H, (45)Ca, and calcium content. Bone turnover as assessed by urinary (3)H-TC was greater in OVX + 1 mo compared to OVX + 3 mo rats up to 6 months postdose. (45)Ca labeling efficiency (% dose/g Ca) was significantly higher than for (3)H and labeling was higher in trabecular-rich than cortical-rich bone. This study affirms that a single administration of either (3)H-TC or (45)Ca is a useful approach to measuring bone turnover directly. The amount of label incorporation into bone was greater in bone sites that were more metabolically active and in all sites when closer vs farther from OVX.