A human skin equivalent was prepared by culturing human keratinocytes on the surface of nylon filtration meshes containing human skin fibroblasts and by growing the epidermal cells at the air-liquid interface. This human skin equivalent model was used to mimic the photoproduction of vitamin D3 in human skin. It was found that the concentration of 7-dehydrocholesterol and its photoconversion to previtamin D3 and its subsequent thermal isomerization to vitamin D3 in the human skin equivalent was essentially identical to that of human skin. The 7-dehydrocholesterol content in the skin equivalent and human skin was 2187 +/- 296 and 2352 +/- 320 ng/cm2, respectively. The percentage of the major photoproducts of 7-dehydrocholesterol in the skin equivalent following ultraviolet B radiation (0.5 J/cm2) was 35% pre-vitamin D3, 29% lumisterol, and 6% tachysterol; 30% remained as 7-dehydrocholesterol. Similarly, in human skin they were 36%, 29%, 7%, and 28%, respectively. After incubation at 37 degrees C for 30 min, 11% and 12% of the previtamin D3 had thermally isomerized to vitamin D3 in the skin equivalent and human skin. In conclusion, compared with cultured keratinocytes or fibroblasts, the human skin equivalent model provides a superior in vitro system that better mimics the physiology and biochemistry of the photosynthesis of vitamin D3 in human skin.