Eph receptors and ephrin ligands are cell-cell communication molecules with well-defined roles in cell adhesion, migration, and tissue boundary formation. However, their expression levels in the squamocolumnar epithelial junction region at the distal esophagus are completely unknown. We examined EphB2 and ephrin-B1 localization in the squamocolumnar epithelial junction region between the proximal and distal stomach of the rodents. Immunostaining showed complimentary expression patterns along the proximal-to-distal axis of the gastric epithelia across the junction: EphB2 expression was maximal around the epithelial junction and sharply decreased in the stratified squamous epithelium at a short distance from the junction, whereas ephrin-B1 was strongly expressed in the stratified squamous epithelium at a distance from the junction and sharply decreased toward the junction. These expression patterns suggest that EphB2/ephrin-B1 signaling occurs preferentially in the epithelia across the junction, where the receptor and ligand expression highly overlap. We also show that (1) EphB2 preferentially binds ephrin-B1, and (2) cell repulsion/lateral migration was induced in primary cultured gastric keratinocytes on ephrin-B1-Fc- and EphB2-Fc-coated surfaces. On the basis of these findings, we propose that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction. © 2013 Springer-Verlag Berlin Heidelberg.