The recruitment of immature dendritic cells into the epidermis is a key step in the development of cutaneous immunity, although the mechanism remains to be clarified. Recently, it was reported that both macrophage inflammatory protein 3alpha (MIP-3alpha)/CCL20 produced by keratinocytes and TNF-alpha are important in recruiting Langerhans cells (LC) to the epidermis. In this study, we examined the production of MIP-3alpha by human keratinocytes stimulated with TNF-alpha. Cultured keratinocytes showed enhanced expression of MIP-3alpha mRNA and protein when stimulated with TNF-alpha. In addition, conditioned medium from TNF-alpha-stimulated keratinocyte cultures induced the migration of L1.2 cells expressing CCR6. We next examined the production of MIP-3alpha in stratified keratinocytes and found that, in contrast to non-stratified keratinocytes, stimulation with TNF-alpha increased the expression of MIP-3alpha mRNA and protein. Moreover, skin samples grown in organ culture and treated with TNF-alpha showed MIP-3alpha in the keratinocytes of the spinous layer, but not in the basal layer, by immunofluorescence staining. Based on these results, we postulate that MIP-3alpha produced by keratinocytes in the spinous layer in response to TNF-alpha stimulation is a key chemokine responsible for the epidermal recruitment of Langerhans cells.