Chromatographic and non-chromatographic procedures in the assay of serum 25-hydroxyvitamin D are compared. Samples extracted with chloroform/methanol were analysed using a competitive protein binding assay, with or without prior chromatography. Non-chromatographic methods constantly gave higher values than chromatographic methods. It is concluded that chromatography is an essential step in the preparation of extracts for the assay of 25-hydroxyvitamin D.