A novel and precise method for determining free and esterified 7-dehydrocholesterol in rat skin was developed by using a two-step high-performance liquid chromatography system. This method involves initial lipid extraction, separation of the free and esterified 7-dehydrocholesterol by, firstly, preparative high-performance liquid chromatography, saponification, and determination by, secondly, analytical high-performance liquid chromatography. This method was applied to the study of the 7-dehydrocholesterol in rat skin. Almost all of the 7-dehydrocholesterol in rat skin was present in an esterified form (78-93%). Ultraviolet exposure of the skin increased total 7-dehydrocholesterol content, however, the percentage of esterified 7-dehydrocholesterol was not altered appreciably. Application of this method to the study of the photo-metabolism of 7-dehydrocholesterol in rat skin revealed that almost all of the vitamin D3 produced by ultraviolet rays was in the esterified form.