We describe a simplified assay for 24,25-and 1.25-dihydroxyvitamin D in human serum. It involves two preparative steps, and normal chick intestine is used in preparing cytosol-binding protein. Our results for 24,25-dihydroxyvitamin D include a reference interval of 2.9—16 nmol/L (1.2—6.7 microgram/L), a mean of 6.7 nmol/L (2.8 microgram/L), an intra-assay CV of 11%, and an interassay CV of 22%. For 1,25-dihydroxyvitamin D, these data were 29—168 pmol/L (12—70 ng/L), 86 pmol/L (36 ng/L), 12%, and 22%, respectively. In hypoparathyroid patients with vitamin D intoxication, mean concentrations of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D in serum were significantly above normal; the 1,25-dihydroxyvitamin D concentrations were significantly below normal. Patients with malabsorption and/or post-gastrectomy states had significantly subnormal values for both 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D in serum, and there was a significantly negative correlation between each of these biochemical values and the severity of osteomalacia. We also discuss cost effectiveness of assaying vitamin D metabolites in human serum.