The in vivo effect of PTH on renal 24-hydroxylase activity of 25-hydroxyvitamin D3 (25OHD3) was examined in vitamin D-deficient thyroparathyroidectomized rats by a recently developed sensitive in vitro assay of 25OHD3-hydroxylases using rat kidney homogenates and by an in vivo assay measuring the accumulation of tritiated metabolites in plasma 5 h after injection of 25OH[3H]D3. Infusion for 20 h of either human PTH-(1-34) (except at 800 pmol/h) or cAMP did not significantly change the plasma levels of calcium and phosphorus compared with those in thyroparathyroidectomized rats given 125 ng 1,25-dihydroxyvitamin D3 to induce renal 24-hydroxylase activity. On the other hand, human PTH-(1-34) markedly inhibited renal 24-hydroxylase activity and stimulated 1-hydroxylase activity. The effective concentration of human PTH-(1-34) was much lower for inhibiting 24-hydroxylase than for stimulating 1-hydroxylase activity. Infusion of less than 100 nmol/h cAMP similarly inhibited 24-hydroxylase activity without enhancing 1-hydroxylase activity. Either theophylline (1.0 mumol/h) or a submaximal dose (25 pmol/h) of human PTH-(1-34) alone inhibited 24-hydroxylase activity only slightly, but the concomitant infusion of both chemicals markedly inhibited 24-hydroxylase activity without stimulating 1-hydroxylase activity. These effects of human PTH-(1-34) and cAMP occurred similarly in both the in vitro and the in vivo assays. The present study clearly indicates that besides its well known action in stimulating 1-hydroxylase activity, PTH inhibits renal 25OHD3-24-hydroxylase activity by a mechanism involving cAMP.