A method is described which permits the assay of 1,25dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 M NhHPO,, pH 10.5. The final purification step involves high-pressure liquid chromatography on PPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%. the purified extract is assayed for 1,25dihydroxyvitamin
D by determining its effect on the resorption of fetal rat bones in vitro. 1,25Dihydroxyvitamin D, standards in the 4-to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r2 of 0.986. Repeat assays with normal human plasma gave a coefficient of variation (SD/i) between experiments of 16.1 f 0.3%. Normal human serum contains 33.4 * 6.4 pgiml (X + SD) 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients. ' Abbreviations used: 1,25-(OH),D, 1,25-dihydroxyvitamin D; hplc, high-pressure liquid chromatography; Tes, 2-{[2-hydroxy-1, I-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid.