Renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) enzyme activity in rats is known to be increased by parathyroid hormone (PTH), hypophosphatemia, and hypocalcemia. Thus, enzyme activity is markedly increased in vitamin D-deficient states, but whether this stimulation is a direct response to the vitamin D deficiency or only occurs following the associated changes in plasma calcium, phosphate, or PTH is unclear. We tested whether vitamin D deficiency per se influences 1-hydroxylase activity in renal cortical slices using a normocalcemic rat model of vitamin D deficiency. Weanling male rats were fed one of the following three diets: (A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D3/g; or vitamin D-deficient diets containing, (B) 0.8% Ca, 0.5% P; and (C) 2.0% Ca, 1.25% P, 20% lactose. Vitamin D-deficient rats fed diet B were hypocalcemic with elevated PTH at both test periods, and 1-hydroxylase activity was increased more than 100-fold compared with rats fed diet A. Plasma calcium, phosphate, and PTH levels were the same in groups A and C, but 1-hydroxylase activity was also substantially elevated in group C versus group A rats (104- and 17-fold increases after 10 and 19 diet weeks, respectively). These data lead to the important conclusion that severe deficiency of vitamin D metabolites per se provides a strong and independent stimulus to renal 1-hydroxylase activity in rats, perhaps due to the absence of 1,25(OH)2D3-mediated enzyme inhibition.