Development and homeostasis of the vascular system requires integrin-facilitated cellular adhesion, migration, proliferation and survival. A specific role for the alpha6beta4 integrin in the vasculature, however, has not been identified. Using immunohistochemistry, we observed alpha6beta4 expression on the dermal microvasculature of human foreskin. Analysis of individual cells isolated from trypsin-disrupted foreskin tissue indicated that alpha6beta4 was expressed by a subset of epithelial and endothelial cells, and not by smooth muscle cells. Expression of alpha6beta4 was also analyzed during new vessel growth using explants of human saphenous vein cultured in fibrinogen gels. The results indicate that alpha6beta4 is not expressed by outgrowing endothelial cells, and is downregulated by the original alpha6beta4-positive endothelial cells of the explant. To determine whether alpha6beta4 is expressed during angiogenesis in vivo, the expression of the beta4 subunit was analyzed during the development of the mouse mystacial (whisker) pad. Immunohistochemical staining of the whisker pad indicates that beta4 is expressed by the adult vasculature. To identify when and where beta4 is turned on in the vasculature, we examined the whisker pads from the developing embryo (E19.5 pc), and from postnatal days zero (P0), three (P3) and seven (P7) pups. The expression of alpha6beta4 was found to be turned on spatially and temporally from caudal to rostral regions and from the deep to superficial vasculature, correlating with the maturation of the whisker pad and its corresponding vasculature. Together, these findings suggest a potential role for alpha6beta4 as a negative component of the angiogenic switch, whereas expression of alpha6beta4 on the adult vasculature may indicate regions requiring additional adhesive mechanisms.