Abstract HIV infection is characterized by high-level replication in gut-associated lymphoid tissues (GALT). This occurs in part because α4β7 +/CD4+ T cells are a preferred target of HIV infection. α4β7 functions as a gut homing receptor. Homing is mediated through a specific interaction with MAdCAM that is expressed on the surface of the high-endothelial venules that line the gut. In addition, α4β7 binds to the V2 domain of HIV gp120. In an SIV model of HIV mucosal transmission, an antibody specific for α4β7 +was found to prevent infection, but the underlying mechanism for this protection is not fully understood. We have proposed that signaling through α4β7 + facilitates HIV transmission, and that the protection from infection that we observed may reflect the capacity of the anti-α4β7 mAb we employed to interfere with this signaling. In order to investigate the role of α4β7+ signaling in mucosal transmission, we have employed a newly developed adhesion assay (Peachman and Rao) that measures the interaction between either the V2 domain of HIV gp120 or MAdCAM with cells expressing α4β7. We have used this assay to evaluate small molecules and antibody antagonists of these interactions. We have found that small molecule antagonists that bind to the active site in α4β7 abrogate binding of cells to both MAdCAM and the V2 domain of HIV gp120. In addition, we determined that retinoic acid increases the adhesion of MAdCAM and V2 to α4β7 expressing cells. This assay should aid in the development of novel strategies designed to interfere with the trafficking of α4β7 +cells to GALT and to mucosal transmission of HIV.