Hyp-mice exhibit abnormal regulation of 25-hydroxyvitamin D [25(OH)D]-1alpha-hydroxylase activity. Previous observations suggest such aberrant modulation is posttranscriptional. To investigate this possibility further, we examined whether hyp-mice manifest abnormal translation of 25(OH)D-1alpha-hydroxylase mRNA. We compared phosphate, parathyroid, and calcitonin effects on renal 25(OH)D-1alpha-hydroxylase protein as well as mRNA and enzyme activity in normal and hyp-mice. We assayed protein by Western blots, mRNA by real-time RT-PCR, and enzyme activity by measuring 1,25-dihydroxyvitamin D production. Although phosphate-depleted mice exhibited enhanced enzyme function, with significantly increased mRNA and protein expression, hyp-mice comparably increased mRNA but failed to augment enzyme activity, concordant with an inability to increase protein expression. PTH stimulation increased mRNA and protein expression as well as enzyme activity in normal mice but in hyp-mice, despite effecting mRNA enhancement, did not increment enzyme function or protein. The inability of hypophosphatemia and PTH to increase 25(OH)D-1alpha-hydroxylase activity and protein expression in hyp-mice was not universal because calcitonin stimulation was normal, suggesting proximal convoluted tubule localization of the defect. These data, in accord with absent undue enhancement of protein expression in hyp-mice treated with protease inhibitors, establish that abberrant regulation of vitamin D metabolism results from abnormal translational activity.