The family of human T cell activation-associated proteins, named VLA complex, is formed by the molecular association of cell surface glycoproteins of 210, 165, 130 and 80 kDa. In this report, we describe eight different monoclonal antibodies (HP mAb) specific for the 80-kDa polypeptide which preferentially associates with the 165-kDa member. Comparative immunoprecipitation and cell-binding studies demonstrated that the HP mAb recognize an epitope(s) on the 165/80 kDa subset different from those recognized by other anti-VLA mAb previously described. Furthermore, cellular and tissue distribution studies by flow cytometry and peroxidase staining showed that the HP reactivity pattern is different from other VLA specificities. The 165/80-kDa complex defined by HP mAb is present on human thymocytes, peripheral blood lymphocytes as well as on T, B and myelomonocytic cell lines. However, only the 80-kDa subunit was precipitated by HP mAb from activated T lymphocytes. These results suggest that the association between the 165- and 80-kDa subunits diminishes during the activation process, and that the epitopes recognized by the HP mAb are located on the 80-kDa protein. The novel polypeptide association of 165/80 kDa has been termed VLA-3.