Involucrin is a marker of keratinocyte terminal differentiation and is expressed only in the suprabasal layers of stratified squamous epithelium. In a previous study with various cell types in culture, we noted that expression of the putative human involucrin promoter was keratinocyte specific. To determine if this promoter is sufficient to direct expression to the suprabasal cells of stratified squamous epithelia in vivo, we have now generated transgenic mouse lines harboring the involucrin promoter sequences linked to a beta-galactosidase reporter gene. In the resulting lines, beta-galactosidase was expressed in the suprabasal compartment of stratified squamous epithelia and in hair follicles in a tissue-specific manner. In the palate, distinct vertical stacks of beta-galactosidase-expressing cells were present, suggesting movement of clonally derived cells through the epithelium. The involucrin gene has a single intron upstream of the translational start site, and removal of this intron did not affect tissue- or stratum-specific expression. These results show that the 3.7-kb involucrin upstream sequences contain all the information necessary for a high level of tissue- and stratum-specific expression.