We describe a precise and specific method for measuring 25-hydroxyvitamin D3 in 1 ml of human serum. Extraction with chloroform/methanol followed by chromatography on a 0.5 X 2-cm silica gel column yields a sample that is sufficiently free of extraneous material for high-performance liquid chromatography on a column of microporous silica gel (10 micron average particle diameter). The measurement is not influenced by vitamins D2 or D3, 25-hydroxyvitamin D2, or any of the more hydroxylated metabolites of the vitamin D group. Results by this method correlate well with a competitive protein-binding assay (r = 0.961), but with a negative bias of 6.9 +/- 3.3 microgram/liter. We measured concentrations of 25-hydroxyvitamin D3 in serum drawn during February from 24 persons who were judged normal by physical examinations. The range was 5.5-23.8 microgram/liter (mean, 15.0 +/- 5.2 microgram/liter). The day-to-day CV for the assay was 5.46%.