Abnormal regulation of vitamin D metabolism is a feature of Xlinked hypophosphatemic rickets in man and of the murine homologue of the disease in the hypophosphatemic (Hyp)-mouse.We previously reported that mutant mice have abnormally low renal 25-hydroxyvitamin D-la-hydroxylase (la-hydroxylase) activity for the prevailing degree of hypophosphatemia. To further characterize this defect, we examined whether Hyp-mouse renal la-hydroxylase activity responds normally to other stimulatory and inhibitory controls of enzyme function. We studied stimulation by parathyroid hormone (PTH) using: (a) a calciumdeficient (0.02% Ca) diet to raise endogenous PTH; or (b) 24-h continuous infusion of 0.25 IU/h bovine PTH via osmotic minipump. In both cases enzyme activity of identically treated normal mice increased to greater levels than those attained by Hypmice. The relative inability of PTH to stimulate la-hydroxylase activity is not a function of the hypophosphatemia in the Hypmouse since PTH-infused, phosphate-depleted normal mice sustained a level of enzyme activity greater than that of normal and Hyp-mice. In further studies we investigated inhibition of enzyme activity by using: (a) a calcium-loaded (1.2% Ca) diet to suppress endogenous PTH; or (b) 24-h continuous infusion of 0.2 ng/h 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The la-hydroxylase activity of normal and Hyp-mice was significantly reduced to similar absolute levels following maintenance on the calcium-loaded diet. Further, infusion of 1,25(0H)2D3 caused a comparable reduction of la-hydroxylase activity in normal, Hyp-, and phosphate-depleted normal mice. These observations indicate that the inhibitory control of la-hydroxylase by reduced levels of PTH or increased 1,25(OH)2D3 concentrations is intact in the mutants. However, the inability of PTH and hypophosphatemia to stimulate enzyme activity in a manner analogous to that in normal and phosphate-depleted mice indicates that a generalized defect of la-hydroxylase regulation is manifest in Hyp-mice.