Renal mitochondria from mutant hypophosphatemic male mice (Hyp/Y) fed a vitamin D-deficient, low calcium diet synthesize significantly less 1,25-dihydroxyvitamin D3 than mitochondria from normal male (+/Y) littermates on the same diet. Kinetic studies reveal that maximum velocity (Vmax) for 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) is lower in Hyp/Y relative to +/Y mice (0.21 +/- 0.02 vs. 1.06 +/- 0.12 pmol/mg protein X min) whereas the apparent Michaelis-Menten constant (Km) for the reaction is not different in both genotypes (0.55 +/- 0.05 vs. 0.50 +/- 0.08 microM). The presence of an inhibitor for 1-hydroxylase activity in renal mitochondria of Hyp/Y mice was ruled out by estimating enzyme activity in mixtures of renal mitochondria from +/Y and Hyp/Y mice. Phosphate in the incubation medium stimulated 1-hydroxylase activity in +/Y mitochondria. In Hyp/Y mice, the stimulation achieved was smaller in magnitude and the added phosphate did not restore mutant 1-hydroxylase activity to normal. The vitamin D-deficient, low calcium diet led to a significant and comparable increase in serum PTH and urinary excretion of cAMP in +/Y and Hyp/Y, suggesting that the mutant strain had an appropriate PTH response to the diet-induced fall in serum calcium. Furthermore, the fractional excretion index of phosphate which is significantly greater in Hyp/Y than +/Y mice fed the control diet increased 3-fold in both genotypes fed the vitamin D-deficient, low calcium diet. These results suggest that the abnormal renal 1-hydroxylase response in Hyp mice is not the result of generalized renal resistance to PTH in the mutant strain and suggest that the defect in Hyp/Y mice may reside at a regulatory step subsequent to cAMP production.