1,25-Dihydroxyvitamin D-24-hydroxylase (24-hydroxylase) modulates the biological effects of 1,25-dihydroxyvitamin D [1,25-(OH)2D] in tissues. The presence of 24-hydroxylase in intestinal mucosa and the mass of the intestine suggest that the intestine is a major site of catabolism of 1,25-(OH)2D. How intestinal levels of 24-hydroxylase are regulated under various dietary conditions, such as calcium (Ca) or phosphorus (P) restriction, is poorly understood. In a series of trials on weanling and mature rats, the effects of dietary Ca or P restriction were compared with the effects of exogenous 1,25-(OH)2D3 administration on intestinal 24-hydroxylase activity. Exogenous administration of 1,25-(OH)2D3, by single bolus injection or constant infusion, increased intestinal 24-hydroxylase activity significantly. Dietary Ca and P restriction both resulted in increased plasma 1,25-(OH)2D3 concentrations several-fold above control rat values (P less than 0.001) and to levels higher than those achieved by constant infusion of 1.3 ng 1,25-(OH)2D3/h. Dietary Ca restriction increased intestinal 24-hydroxylase 6- to 20-fold above that of rats fed a Ca-replete diet (P less than 0.001). Dietary P restriction had no significant effect on intestinal 24-hydroxylase activity. These data suggest that dietary Ca restriction results in increased plasma levels of 1,25-(OH)2D3, which, in turn, leads to up-regulation of intestinal 24-hydroxylase. Conversely, dietary P restriction prevents 1,25-(OH)2D3-mediated up-regulation of 24-hydroxylase.