We describe a method for measuring 25-hydroxyvitamin D in serum. The serum is denatured with ethanol, and the extract purified by chromatography on silicic acid to prevent potential interference from vitamin D and by other substances. 25-Hydoxyvitamin D is assayed by sequential saturation analysis in Tris-HCI buffer, pH 8.6, containing the surfactant Triton X-405 (2 ml/liter) to solubilize the steroid. Diluter, unpurifed normal human serum is used as the binder. The method is sensitive and precise. The dose-response curve is linear. The assay normally gives high count rates (12 000 to 20 000 cpm for the zero standard) and consequently 25-hydroxyvitamin D of low specific activity can be used if necessary. Serum 25-hydroxyvitamin D was determined in normal and osteomalacic subjects.