The human involucrin gene, which encodes a precursor of the keratinocyte cornified layer, is strongly expressed in response to differentiation stimuli. Earlier studies suggested that YY1 and components of the AP-1 family might participate in the silencing of involucrin in proliferating keratinocytes. This study shows that overexpression of either YY1 or c-Jun represses transcription of the human involucrin gene in multiplying keratinocytes. Transient overexpression and site-directed mutagenesis experiments of the involucrin 5'-non-coding region (5'-NCR) confirmed that YY1 and c-Jun repress involucrin transcription. This repression involves the distal zinc fingers of YY1 protein and the DNA binding and leucine zipper domains of c-Jun. The results with protein pull-down experiments are consistent with the hypothesis that interaction of YY1 with c-Jun is an important mechanism for involucrin repression. Cotransfection of YY1 modified the stimulatory function of mutant c-Jun proteins independently of their DNA binding capacity suggesting that interactions may be more complex in vivo. Additionally, c-Jun protein levels are affected by differentiation stimuli indicating the importance of c-Jun in the YY1 repression pathway. Thus YY1 and c-Jun have an important role in epidermal differentiation by negatively regulating the human involucrin gene.