Measurement of the serum level of 25-hydroxyvitamin D is the most useful parameter in evaluating vitamin D status. The serum level of vitamin D is a useful parameter in studying short time effects after exposure to ultraviolet light and absorption of the vitamin after oral administration. A method for simultaneous determinations of vitamin D3 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is described. Serum or plasma was extracted by methanol-isopropanol (90:10, v/v) and hexane. The hexane layer was injected in to a reversed-phase (C18) high-performance liquid chromatographic (HPLC) system. 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were eluted by methanol-water (85:15, v/v), and vitamin D3 by a linear gradient of methanol-water (85:15) and methanol-isopropanol-water (87.5:10:2.5), and detected by u.v. absorption. This method gave separate determinations of the D2 and D3 forms of 25-hydroxyvitamin D, but owing to an interfering peak the method does not measure vitamin D2. The assay was very sensitive with a detection limit of 5 nmol l-1 for 25-hydroxyvitamin D2 and D3 and vitamin D3 by using 0.5 ml serum or plasma for analysis, so that for low vitamin D3 levels more than 1 ml of serum is desirable.