The alpha 4 integrin subunit can associate with either the beta 1- or beta 7-integrin subunit to form two unique adhesion receptors alpha 4 beta 1 and alpha 4 beta 7. We developed a monoclonal antibody (mAb 19H8) that immunoprecipitated alpha 4 beta 1, induced homotypic leukocyte aggregation, and blocked the binding of cells to a synthetic peptide corresponding to the CS-1 peptide region of fibronectin. Aggregation cross-blocking analysis suggested that mAb 19H8 belonged to the group of mAbs that react with the B2 epitope of the alpha 4 subunit (alpha 4.B2 epitope); however, unlike the alpha 4.B2-specific mAb L25, mAb 19H8 did not immunoprecipitate alpha 4 beta 7. In addition, mAb 19H8 did not bind to beta 1-positive cells unless transfected with alpha 4 cDNA. These results indicated that mAb 19H8 was not specific for an individual alpha 4, beta 1, or beta 7 subunit but reacted with an epitope formed from the association of alpha 4 with beta 1. Separating the alpha 4 from the beta 1 subunit, by removing divalent cations or by treatment with high pH, disrupted mAb 19H8 binding. In contrast, the alpha 4-specific mAb L25 and the beta 1-specific mAb 18D3 could react with their respective subunits without subunit association. Therefore, mAb 19H8 defined a novel regulatory epitope expressed by the integrin alpha 4 beta 1.