A single column reverse phase high pressure liquid chromatographic method is described for the determination of vitamins D2 and D3 in fluid milk. Resolution of vitamin D2 from D3 is helpful for use as an internal standard. The method involves overnight saponification at room temperature, extraction of unsaponifiables, precipitation of cholesterol, and aluminum oxide column cleanup. Sample extracts were chromatographed under isocratic conditions on a 10 micron Vydac reverse phase column using acetonitrile-methanol (90 + 10) as the mobile phase. In addition, a MicroPak MCH-5 reverse phase column with acetonitrile as the mobile phase was used with an automatic system for one product type. Thirty samples each of homogenized (3.8% fat), low fat (2.0% fat), and skim (less than or equal to 0.5% fat) milk spiked with 200, 400, and 600 IU vitamin D/qt were analyzed. Coefficient of variation (CV) and percent recovery for each product type and each spike level of vitamins D2 and D3 were calculated from 10 replicate analyses. Vitamin D2 recoveries for all product types at the 3 fortification levels varied from 85.2 to 99.7%; vitamin D3 recoveries varied from 85.9 to 98.8%. The minimum detectable quantity of vitamin D in milk was 15 IU/qt.