During wound healing, cell-cell interactions between epidermal keratinocytes and dermal fibroblasts contribute to the organization of epidermis, in which prostaglandin E2 (PGE2) is considered to be involved in proliferation and differentiation of keratinocytes. In the current study, we investigated the regulation of PGE2 biosynthesis in co-culture of human epidermal keratinocytes and human dermal fibroblasts. The production of PGE2 was synergistically enhanced in the co-culture at cell ratios of keratinocytes to fibroblasts between 1/8 and 4, whereas the production of PGE2 was negligible in individual monolayer cultures of keratinocytes or fibroblasts. To address the mechanism of PGE2 production induced by the co-culture of keratinocytes and fibroblasts, we asked whether either cell-derived soluble factor(s) or direct cell-cell contact was required to augment the production of PGE2. Neither the fibroblast-conditioned medium nor membrane fractions influenced the production of PGE2 in keratinocytes. Keratinocyte-conditioned medium greatly enhanced the production of PGE2 in fibroblasts, however, whereas the effect of keratinocyte-membrane fractions was weaker. The main soluble fraction in the keratinocyte-conditioned medium contained a precursor of interleukin-1 alpha (proIL-1alpha) by western blot analysis, and PGE2 production was inhibited by anti-IL-1alpha antibody, but not by anti-IL-1beta or by anti-tumor necrosis factor-alpha antibody. The enhanced production of PGE2 in fibroblasts upon culturing with keratinocytes was due to the induction of COX-2 mRNA mediated by proIL-1alpha released from keratinocytes. These results suggest that cell-cell interactions of keratinocytes and fibroblasts augment the production of PGE2 by a mechanism in which the activity of COX-2 in fibroblasts is increased by the keratinocyte-derived proIL-1alpha in a paracrine manner.