Keratinocyte growth factor (KGF) and alpha(5)beta(1)-integrin are not expressed in normal skin but they are both highly upregulated in the migrating epidermis during wound healing. Here we report that KGF increased alpha(5) mRNA and protein levels in epidermoid carcinoma cells and stratified bioengineered epidermis. Interestingly, KGF increased integrin alpha(5) in the basal as well as suprabasal cell epidermal layers. Promoter studies indicated that KGF-induced integrin alpha(5) promoter activation was dependent on the C/EBP transcription factor binding site. Accordingly, KGF induced sustained phosphorylation of C/EBP-beta that was dependent on activation of ERK1/2. In addition, a dominant negative form of C/EBP-beta inhibited alpha(5) promoter activity and blocking C/EBP-beta with siRNA diminished integrin alpha(5) expression. Taken together, our data indicate that KGF increased integrin alpha(5) expression by phosphorylating C/EBP-beta. Interestingly, KGF-induced upregulation of integrin alpha(5) was more pronounced in three-dimensional tissue analogues than in conventional two-dimensional culture suggesting that stratified epidermis may be useful in understanding the effects of growth factors in the local tissue microenvironment.