Vitamin D was determined in fortified milk by high pressure liquid chromatography (HPLC) after preliminary purification involving saponification at room temperature and low pressure chromatography on hydroxyalkoxyprophyl Sephadex (HAPS). The saponified milk (50 ml) was extracted with ether, the extract was chromatographed in hexane on a 60 X 1 cm column of HAPS, and the absorption of the eluate at 254 nm was recorded. The fraction containing vitamin D was collected by using the position of characteristic peaks in the chromatogram as a guide. An aliquot of this fraction was chromatographed in an HPLC system on a 25 cm X 3.2 mm column of LiChorosorb Si 60 5 micrometer silicic acid, using 0.6% isopropanol in hexane (50% water-saturated) as the mobile phase. The absorption was set at 265 nm. Vitamin D was cleanly separated from other absorbing materials, and the amount could be calculated from the peak area. Recovery of a added vitamin D was greater than 97%.