The skin develops and differentiates during embryogenesis, which is concertedly regulated by a variety of genes. The present study isolated from the rat embryonic skin a novel differentiation-associated gene named Kdap (keratinocyte differentiation-associated protein) by suppression subtractive hybridization between the skin of 14day postcoitus (dpc) embryo (the prehair-germ stage) and that of 17dpc embryo (the hair-germ stage). Its mRNA contained four spliced forms in these tissues. The gene encoded a protein of total 98 amino acids with a calculated molecular mass of 11kDa and an isoelectric point of 6.1 as an unspliced form. The two splicing zones were well conserved among rat, mouse, and human. This protein had a high hydrophobic N-terminal region, a possible signal sequence, and contained two putative N-myristoylation sites and two casein kinase II phosphorylation sites. In situ hybridization experiments detected Kdap transcripts exclusively in the suprabasal cell layers of the embryonic epidermis. Intense expression was also seen in suprabasal cells in regions of infundibulum of the hair follicle. These results indicated that Kdap provides a new insight into the mechanism of differentiation and the maintenance of stratified epithelia.