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