24-Hydroxylase is found in many mammalian tissues and is required as an initial step in the deactivation of vitamin D3 metabolites and most of its active analogs. We studied the regulation of intestinal 24-hydroxylase (I-24-OHase) activity and messenger RNA (mRNA) expression in rats as influenced by calcium and vitamin D status. Rats were fed vitamin D-replete diets containing either normal calcium (1.0-1.2%, designated NC) or low calcium (0.02%; designated LC). Half of the NC and LC rats received 25,000 IU vitamin D3 three times weekly, orally, and were designated NCT and LCT, respectively. We found that I-24-OHase mRNA expression was up-regulated in rats receiving excess vitamin D3 (NCT and LCT). We observed, however, that the up-regulation was much more dramatic in the LCT group and exceeded by 4.5-fold that observed in the NCT group. Plasma calcium was also elevated in the NCT group (12.6 +/- 0.2 mg/dl), but not the LCT group (10.5 +/- 0.15 mg/dl). We, therefore, examined the possibility that calcitonin released in response to hypercalcemia may have suppressed the induced expression of I-24-OHase mRNA in the NCT group. The plasma calcitonin level was higher in the NCT group (36.14 +/- 2.46 pg/ml) relative to that in the LCT group (19.38 +/- 2.28 pg/ml). Thyroparathyroidectomy also resulted in a 2-fold (P < 0.001) increase in I-24-OHase activity in the NCT group, a response that was reversed (within 4 h) with a single dose of calcitonin (100 IU/rat). Calcitonin administration to LCT rats also resulted in a significant (P < 0.001) 5-fold reduction in I-24-OHase mRNA expression. These data suggest that calcitonin is a potent negative regulator of I-24-OHase mRNA expression and I-24-OHase activity and that the release of calcitonin may block an important pathway for the inactivation of vitamin D3 metabolites in intestine and, thereby, potentiate the toxicity of vitamin D3 during periods of its excess consumption.