Vitamin D intoxication was produced with oral doses of either vitamin D₃ or 25-hydroxyvitamin D₃ in CYP27B1 -/- (1α-hydroxylase knockout) and wild-type mice. These compounds were equally toxic in wild-type and the mutant mice. Since the null mutant mice are unable to produce 1,25-dihydroxyvitamin D, it is clear 1,25-dihydroxyvitamin D is not responsible for vitamin D intoxication. On the other hand, 25-hydroxyvitamin D rises to levels of 400-700 ng/ml or 1000-1750 nM in the serum of both groups of mice. Toxicity was evidenced by severe hypercalcemia and weight loss. Measurement of 1,25-dihydroxyvitamin D₃ in serum confirmed its absence from serum of the CYP27B1 -/- mice given 25-hydroxyvitamin D₃. Since high concentrations of 25-hydroxyvitamin D can bind the vitamin D receptor and can induce transcription, 25-hydroxyvitamin D is likely responsible for toxicity of vitamin D excess.