The up-regulation of the 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, the mechanism of down-regulation of the 24-hydroxylase by parathyroid hormone (PTH) has not yet been elucidated. To study the mechanism of PTH action, we used AOK-B50 cells, a porcine kidney-cell line with stably transfected opossum PTH receptor in which both the 24-hydroxylase mRNA and activity are down-regulated by PTH. Cells dosed with 1,25(OH)2D3 at 0 h, and subsequently at 0, 1, 2, or 4 h with 100 nM of PTH, showed levels of 24-hydroxylase mRNA equivalent to 72.6, 65.3, 57.2, and 37.1%, respectively, of the levels found in cells dosed with 1,25(OH)2D3 only. All cells were collected 7 h after the initial 1,25(OH)2D3 dose. This pattern of expression indicated that PTH does not act by repressing transcription but rather by making the mRNA for 24-hydroxylase susceptible to degradation. At least 1 h is required for PTH to act. Further RNA and protein syntheses are required for PTH to act. However, the sites and mechanism whereby PTH causes 24-hydroxylase mRNA degradation are unknown. Because the untranslated regions of genes can determine the stability of its transcripts, we studied the 5 untranslated region and the 3 untranslated region of the rat 24-hydroxylase gene by using reporter-gene strategy to identify possible PTH sites of action. None was found, suggesting that the destabilization site is elsewhere in the coding region.calcium metabolism ͉ vitamin D endocrine system V itamin D, as synthesized in the skin or ingested in the diet, is sequentially hydroxylated at the C25 position in the liver and at the C1␣ position in the kidney to form the active metabolite 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] (1). 24-Hydroxylase is the enzyme responsible for the first step in the catabolism of 1,25(OH) 2 D 3 that ultimately leads to the excretion of the hormone as calcitroic acid (1). The 1␣-hydroxylase and the 24-hydroxylase are very tightly and reciprocally regulated by 1,25(OH) 2 D 3 itself and parathyroid hormone (PTH) (2). 1,25(OH) 2 D 3 activates its own breakdown by strongly inducing the 24-hydroxylase, while at the same time down-regulating the 1␣-hydroxylase (1). PTH induces the 1␣-hydroxylase while down-regulating the 24-hydroxylase. The induction of 24-hydroxylase mRNA by 1,25(OH) 2 D 3 has been extensively studied and is largely due to the actions of the vitamin D receptor complexes on two vitamin D response elements in the promoter of the 24-hydroxylase gene (3, 4). The mechanism of downregulation by PTH remains to be elucidated. It has been shown that PTH acts to decrease 24-hydroxylase mRNA, but it remains unknown whether the decrease is caused by changes in the transcription mechanism or whether it is caused by effects of PTH on mRNA stability (5). By using AOK-B50 cells, a porcinecell line with stably transfected opossum PTH receptors (6), we have found th...