The vitamin D(3)-25-hydroxylase CYP27A is located predominantly in liver, but its expression is also detected in extrahepatic tissues. Our aim was to evaluate the regulation of CYP27A by vitamin D(3) (D(3)) or its metabolites in rat duodena. Vitamin D-depleted rats were repleted with D(3), 25-hydroxyvitamin D (25OHD), or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or acutely injected 1,25(OH)(2)D(3) to investigate the mechanisms of action of the hormone. All D(3) compounds led to a progressive decrease in CYP27A mRNA, with levels after D(3) representing 20% of that observed in D depletion. 25OHD decreased CYP27A mRNA by 55%, whereas 1,25(OH)(2)D(3) led to a 40% decrease, which was accompanied by a 31% decrease in CYP27A protein levels and an 89% decrease in enzyme activity. Peak circulating 1,25(OH)(2)D(3) concentrations were, however, the highest in D(3)-repleted, followed by 25OHD- and 1,25(OH)(2)D(3)-repleted animals. 1,25(OH)(2)D(3) resulted in a decrease in both CYP27A mRNA half-life and transcription rate. Our data illustrate that the intestine expresses the D(3)-25-hydroxylase and that the gene is highly regulated in vivo through a direct action of 1,25(OH)(2)D(3) or through the local production of D(3) metabolites.