We studied the effects of dietary calcium (Ca) restriction and excess vitamin D3 on tissue 25-hydroxyvitamin D-1-hydroxylase (1-OHase) and 1,25(OH)2D/25-OH-D-24-hydroxylase (24-OHase) activities in rats. Effects were studied in four groups of rats, with each group receiving one of the following diets: a control diet consisting of normal Ca and normal vitamin D3 (NC), NC plus excess (75,000 IU/week) vitamin D3 (NCT), low Ca and normal vitamin D3 (LC), or LC diet with excess vitamin D3 (LCT). Rats fed the low-Ca diets (LC and LCT) had elevated plasma parathyroid hormone (PTH) concentrations, increasing > 3-fold relative to rats fed the normal Ca diets. The elevated concentrations of PTH in LCT rats did not result in increased plasma 1,25-dihydroxycholecalciferol [1,25(OH)2D3] (NC = 115 +/- 7 pg/ml; LCT = 99 +/- 11 pg/ml). Plasma 1,25(OH)2D in LC rats, however, was increased significantly (615 +/- 110, P = < 0.001). There were no differences in either plasma Ca or phosphorus between the LC and LCT groups. Dietary Ca restriction led to an 18-fold stimulation in renal 1-OHase activity in LC rats (P = < 0.01), while 1-OHase in the LCT rats was marginally but significantly elevated 2.3-fold (P = < 0.05). The ability of PTH to downregulate renal 24-OHase and the 1,25-dihydroxyvitamin D receptor (VDR) during prolonged Ca restriction remained intact, irrespective of vitamin D status. Also, the metabolic clearance rate for 1,25(OH)2D3 was enhanced by feeding excess vitamin D3, which was likely a result of the substantial elevations in intestinal (25-fold) and renal (46-fold) 24-OHase activities in the LCT and NCT groups, respectively. These data indicate that calcium restriction accompanied by excess vitamin D3 is attended by impaired responsiveness of renal 1-OHase to PTH and enhanced metabolic clearance of 1,25(OH)2D.