Using 2 experiments, a new method was established to determine 7-dehydrocholesterol [7-DHC], previtamin D3 [pre-D3] and vitamin D3 in rat skin. The 7-DHC was photochemically converted into vitamin D3 by UV irradiation. The in vivo conversion mechanism might be similar to the in vitro one because there were little differences between the corresponding estimated values of vitamin D3 in the 2 experiments. The peak due to pre-D3 was not observed in the HPLC [high pressure liquid chromatography] chromatograms of the 2 experiments. Since the presently proposed method included the saponification procedure with refluxing to eliminate interfering substances, the produced pre-D3 might be thermally converted into vitamin D3 during the procedure and the mono peak of vitamin D3 alone was observed in each HPLC chromatogram.