The integrin beta 8 subunit was identified by cloning and sequencing of the cDNA and has been shown to associate with the alpha v subunit (Moyle, M., Napier, M. A., and McLean, J. W. (1991) J. Biol. Chem. 266, 19650-19658). We now present initial data on its functional properties. We produced a recombinant secreted form of alpha v beta 8 and used it to raise monoclonal antibodies that recognize the alpha v beta 8 complex or the beta 8 subunit alone on the surface of melanoma cells and on beta 8-transfected human embryonic kidney (293) cells. Affinity chromatography experiments showed that secreted alpha v beta 8 bound to vitronectin but not to fibronectin, collagen, or fibrinogen. Supporting evidence that intact full-length alpha v beta 8 could also bind to vitronectin-Sepharose was provided by performing affinity chromatography with the melanoma cell line MeWo, which normally expresses the intact beta 8 subunit. By studying the adhesive properties of melanoma cells and beta 8-transfected 293 cells, we found that alpha v beta 8 by itself does not promote cell adhesion on a vitronectin-coated substrate. To test the respective functional activities of the beta 8 extracellular and cytoplasmic domains, we analyzed chimeric beta 8/beta 3 subunit constructs. The beta 3 subunit was chosen because full-length beta 3, when transfected into 293 cells, strongly supports cell adhesion. We found that a chimeric integrin containing the beta 3 extracellular domain combined with the beta 8 transmembrane and cytoplasmic domains did not promote 293 cell adhesion. Conversely, a chimeric integrin construct combining the beta 8 extracellular domain with the beta 3 transmembrane and cytoplasmic domains did promote adhesion of transfected 293 cells. This suggests that the beta 8 cytoplasmic domain does not interact with the cytoskeleton and with cytoplasmic signaling pathways in an adhesion-promoting fashion. We conclude that the beta 8 cytoplasmic domain, which is structurally unrelated to the conserved cytoplasmic domains of other beta subunits, is functionally distinct.