The transcription factor AP-2/promoter system is essential for gene expression associated with ectodermal development, particularly in the neural crest and skin. Three AP-2 isoforms, alpha, beta, and gamma, exhibit a highly homologous structure, but their functions are considered to be different. Here, we report on the role of each AP-2 isoform in complex keratinocyte biology including proliferation, differentiation, and carcinogenesis. The expression of AP-2 was investigated immunohistochemically in serial skin sections from normal and psoriatic skin, and squamous cell carcinoma (SCC). AP-2alpha was present only in the nuclei of normal basal keratinocytes, but was significantly increased in lesional proliferating keratinocytes of both diseases. AP-2beta was completely absent from all skin samples except dermal sweat glands, whereas AP-2gamma was present homogeneously throughout the epidermis in normal and psoriatic skin as well as in the SCC lesion. Their restricted expression patterns correlated with in vitro DNA binding assays using selective keratinocyte gene promoters and three recombinant AP-2 isoforms generated bacterially as glutathione S-transferase fusion protein. Epidermal growth factor receptor and basal keratin K14 promoters bound to AP-2alpha and AP-2gamma with similar affinities, whereas suprabasal keratin K1, type I transglutaminase, and involucrin promoters predominantly bound to AP-2gamma rather than AP-2alpha. In contrast, AP-2beta did not bind to any of the five promoters despite specific binding to the AP-2 consensus probe. These results suggest that AP-2alpha is closely associated with keratinocyte proliferation and/or carcinogenesis rather than differentiation, while AP-2gamma is ubiquitous in all stages of keratinocyte biology. Taken together, three AP-2 isoforms perform unique roles in the spatial and temporal expression of human keratinocyte-related genes, thereby maintaining epidermal homeostasis. Disruption of the epidermal AP-2 balance may contribute to hyperproliferative conditions, such as psoriasis and SCC.