25-Hydroxyvitamin D3-1α-hydroxylase activity was assayed in primary serum-free monolayer tissue culture of renal cortical cells from hypophosphatemic (Hyp) mice and normal litter mates. Morphological and growth characteristics of cells from the two genotypes were indistinguishable. Basal enzyme activity was not significantly different in either type of cell over a wide range of substrate concentration. The enzyme from both genotypes was stimulated by PTH and suppressed by increased phosphate concentration in the culture medium. Whereas 1α-hydroxylase activity in cells from normal mice was increased in low calcium medium and suppressed in high calcium medium, the enzyme in cells from Hyp mice was not altered by similar changes in the medium calcium concentration. Salmon calcitonin caused a significant increase in 1α-hydroxylase in cells from normal mice, but did not stimulate enzyme activity in cells from Hyp mice. These studies indicate that control of 1α-hydroxylase activity is abnormal in renal cortical cells from Hyp mice. Impaired control of this enzyme could result in the inappropriately low circulating concentrations of 1,25-dihydroxyvi-tamin D3 that have been observed in humans with hypophos-phatemic rickets and in the relatively low activity of 1α-hydrox-ylase in renal cortical homogenates of Hyp mice compared to that in normal mice on a low phosphate diet.