Identification of vitamin D3 and 7-dehydrocholesterol (7-DHC) in cow's milk by gas chromatography-mass spectrometry (GC-MS) and their quantitation by high-performance liquid chromatography (HPLC) were investigated. When vitamin D and provitamin D fractions purified from a sample of commercial cow's milk were applied to GC-MS, the results showed that the fractions contained vitamin D3 and 7-DHC, respectively, while neither vitamin D2 nor ergosterol could be detected in the milk. HPLC methods for the determination of vitamin D3 and 7-DHC in cow's milk were then proposed as routine methods. The method for assaying vitamin D3 included the isolation of lipids from milk according to the directions of Bell and Christie (5), saponification, isolation of unsaponifiable matter, digitonin-Celite column chromatography, preparative thin-layer chromatography (TLC) and application to HPLC. On the other hand, 7-DHC in milk could be simultaneously determined without the purification by digitonin-Celite column chromatography. The peak corresponding to either vitamin D3 or 7-DHC in the respective HPL-chromatograms was clearly separated from possible interfering substances and the recovery experiments for both vitamin D3 and 7-DHC gave satisfactory results. When the proposed methods were applied to 10 samples of commercial cow's milk, the assayed values of vitamin D3 and 7-DHC were 19--79 I.U./liter and 14--56 microgram/liter, respectively.