Human keratinocytes grown on deepidermized dermis (DED) are able to reconstruct a morphologically normal stratified and keratinized epidermis. This culture system is suitable for studying in vitro the effects of various hormones and factors on epidermal differentiation, and the goal of the present work was to study the effect of vitamin D. We found that the hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3, produced very specific alterations in epidermal architecture in a dose-dependent manner, consisting of significant reduction of the nucleated layers of the epithelium, but not of the stratum corneum, which was instead slightly thickened. The study of stage-specific differentiation markers showed that the two extreme layers of epidermis, i.e. the basal layer and the stratum corneum, were unaffected by the hormone, but that the reduction involved specifically the intermediate differentiation compartment, i.e. the spinous and granular layers. It was shown that the reduction of the intermediate compartment provoked by 1,25-dihydroxyvitamin D3 is not due to a block in the proliferation of basal cells or to inhibition of their differentiation into suprabasal cells, but to stimulation of the terminal differentiation of suprabasal cells into corneocytes.