Glomerular development proceeds through the spatially ordered and sequential recruitment, proliferation, assembly, and differentiation of endothelial, mesangial, and epithelial progenitors. The molecular determinants of cell-cell recognition and targeting in this process have yet to be defined. The Eph/ephrin family of membrane receptors and counter-receptors are critical participants of developmental vascular assembly in extrarenal sites. Renal expression patterns of ephrin-B2 and EphB4 were investigated using mice expressing beta-galactosidase under control of ephrin-B2 or EphB4 promoters. The earliest glomerular expression of ephrin-B2 was identified in a subset of differentiating comma-stage glomerular epithelial cells (podocyte progenitors) adjacent to the vascular cleft where endothelial progenitors are subsequently recruited. Epithelial ephrin-B2 expression was accompanied by expression in endothelial and mesangial cells as capillary assembly progressed. At or near completion of glomerular maturation, epithelial ephrin-B2 expression was extinguished, with persistence in glomerular endothelial cells. Throughout development, one of several ephrin-B2 receptors, EphB4, was persistently and exclusively expressed in endothelial cells of venous structures. The findings show sequential ephrin-B2 expression across glomerular lineages, first in a distinct subset of podocyte progenitors and subsequently in endothelial cells of the developing glomerulus. Given targeting functions for Eph/ephrin family proteins, the findings suggest that ephrin-B2 expression marks podocyte progenitors at the site of vascular cleft formation, where expression may establish an "address" to which endothelial and mesangial progenitors are recruited. Thus, the present results suggest that ephrin-B2 and EphB interactions play an important role in glomerular microvascular assembly.