We report here the isolation of two new monoclonal antibodies (MB1.1 and MB1.2) against mouse VLA-beta 1 integrin subunit. Characterization by flow cytometry demonstrated binding of MB1.1 and MB1.2 to freshly isolated thymocytes, primary bone marrow mast cell lines, as well as cell lines of distinct lineage each expressing different combination of VLA integrins. The specificity of MB1.1 and MB1.2 was determined by (1) their binding to antigen with M(r) about 120 kDa, and (2) the ability of antiserum against the carboxyl terminal of VLA-beta 1 subunit to deplete antigens for MB1.1 and MB1.2 in sequential immunoprecipitation experiments. The epitopes for MB1.1 and MB1.2 were in close proximity to each other since preincubation of cells with one MAb inhibited the binding of the other. However, MB1.1 and MB1.2 differed in their affinity for the beta 1 subunit. In addition, neither MAbs had any effect on cell adhesion to matrix proteins indicating that the epitopes involved are distant from VLA integrin ligand-binding sites. MB1.1 and MB1.2 appear to differ from the two MAbs so far reported against mouse VLA-beta 1 subunit, KMI6 and 9EG7. Thus, the epitopes for MB1.1 and MB1.2 were readily detectable on unfractionated thymocytes whereas KMI6 has been reported to bind only a fraction of CD4-8- and CD4-8+ thymocytes. Phorbol ester and Mn2+, which have been shown to regulate the binding of 9EG7, had no effect on MB1.1 and MB1.2 binding to VLA-beta 1 integrin subunit.