Cell-extracellular matrix (ECM) and cell-cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell-cell contact while maintaining constant cell-ECM areas. Bowtie-shaped micropatterned areas (75-1600 microm(2)) were engineered to either permit or prevent cell-cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell-cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell-cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell-cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell-cell contact regulates early keratinocyte differentiation independently from changes in cell shape.