Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of the skin. The gene expression of this differentiation marker is induced by elevated extracellular calcium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. We have sequenced the upstream region of the involucrin promoter and localized a calcium response element that contains an activating protein-1 (AP-1) site (TGAGTCA). Mutation of this site abolished the promoter activation by calcium. Compared with cells grown in 0.03 mm calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mm calcium. Immunoelectrophoretic mobility shift (supershift) assays identified JunD, Fra1, and Fra2 as the major factors that bind to the AP-1 element. Western analysis of the proteins in the nuclear extracts showed that the levels of c-Jun, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 decreased slightly with calcium treatment. The effect of calcium on the involucrin promoter was enhanced synergistically by phorbol 12-myristate 13-acetate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-regulated involucrin gene expression is mediated at least in part by AP-1 transcription factors.