The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.