CD40 is expressed on a wide array of hematopoietic and non-hematopoietic cells, including keratinocytes. The pivotal in vivo function of CD40 on hematopoietic cells in the regulation of both humoral and cell-mediated immunity is well established. However, whether CD40 expression on non-hematopoietic cells influences immunity has until now not been addressed. Therefore, we transgenically expressed human CD40 (hCD40) under the control of the keratin 14 promoter to drive expression of hCD40 in basal keratinocytes of mice. When we selectively engaged hCD40 in vivo on the keratinocytes of these mice, the keratinocytes secreted TNF-alpha, resulting in dendritic cell migration to draining lymph nodes and enhanced in vitro T cell priming to an epicutaneously applied chemical sensitizer. Exclusive CD40 engagement on keratinocytes during a contact hypersensitivity response displayed exacerbated and prolonged cutaneous immune reactions relative to control mice. Thus, CD40 engagement on non-hematopoietic cells, such as keratinocytes, can amplify cutaneous and regional T cell responses in vivo.