Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogen-activated protein kinase kinase, components of the extracellular signal-regulated protein kinase (ERK) pathway, thereby inhibiting the pathway and resulting in the suppression of cell proliferation. In the present study, we examined whether PEBP is involved in differentiation induction of human keratinocytes. PEBP expression was immunohistochemically examined in normal human skin and skin cancers with different differentiation properties. PEBP was not expressed in the basal layer of the epidermis but was expressed in the spinous and granular layers of normal skin. The protein was expressed in differentiated but not in undifferentiated carcinoma. PEBP expression was also examined in cultured normal human epidermal keratinocytes in which differentiation was induced by calcium treatment. Involucrin was used as a differentiation marker for spinous and granular cells. Northern blotting analysis indicated that both PEBP and involucrin mRNAs were enhanced 6 h after treatment with 2.0 mM CaCl(2). The protein amount of PEBP was also increased by this treatment. To investigate whether PEBP is involved in differentiation induction of keratinocytes, HaCaT keratinocytes were transfected with an expression vector. Fluorescent immunostain revealed that cells expressing PEBP exhibited enlarged and flattened cell shape, and induction of involucrin expression was demonstrated by immunoblot analysis. Although the protein amount of ERK was not altered, phosphorylated ERK levels were decreased and cell proliferation was partly inhibited by PEBP expression. These results indicate that PEBP not only inhibits cell proliferation but also induces differentiation of human keratinocytes.