In order to confirm the photochemical conversion of 7-dehydrocholesterol (7-DHC) into vitamin D₃ in rat skin, the following in vitro and in vivo experiments were carried out. In the first (in vitro) experiment, the skin stripped off from a sacrificed normal rat was irradiated with an ultraviolet (UV) lamp for a constant period. In the second (in vivo) experiment, the normal rat, irradiated under the same condition mentioned above, was sacrificed and then the skin was stripped off. Lipids were individually extracted with chloroform-methanol (1:1) from the skin obtained in the two experiments and the solvent was evaporated. The resulting residue was saponified and the unsaponifiable matter extracted with benzene was purified by application to hydroxyalkoxypropyl (HAP) Sephadex column chromatography. The resulting purified vitamin D₃ fraction was applied to high-performance liquid chromatography (HPLC) in order to estimate vitamin D₃. No peak, aside from that of α-naphthol as an internal standard, was observed in the HPLC chromatogram on the skin obtained from the non-irradiated rat, whereas the peak corresponding to vitamin D₃ was observed in each HPLC chromatogram on both the irradiated skin (in vitro experiment) and the skin obtained from the irradiated rat (in vivo experiment). The peaks, confirmed to be due to vitamin D₃ by the results of co-chromatography, were increased according to the increase of irradiation energy and there were little differences between the corresponding estimated values of vitamin D₃ in the two experiments. These results prompted the conclusion that 7-DHC in rat skin was photochemically converted into vitamin D₃ by UV irradiation and that the in vivo vonversion mechanism might be the same as the in vitro one.