INTRODUCTION: Folate receptor beta (FRß) is only detectable in placenta and limited to some hematopoietic cells of myeloid lineage in healthy people. Studies have indicated that FRß is over-expressed in activated macrophages in autoimmune diseases and some cancer cells. In this study we aimed to develop an FRß-specific human monoclonal antibody (mAb) that could be used as a therapeutic agent to treat rheumatoid arthritis and other autoimmune diseases, as well as FRß positive cancers. METHODS: Functional recombinant FRß protein was produced in insect cells and used as antigen to isolate a mAb, m909, from a human naïve Fab phage display library. Binding of Fab and IgG1 m909 to FRß was measured by ELISA, surface plasmon resonance, immune fluorescence staining, and flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with FRß positive CHO cells as target cells and isolated peripheral blood monocytes as effector cells in an in vitro assay. RESULTS: Fab m909 bound with relatively high affinity (equilibrium dissociation constant 57 nM) to FRß. The IgG1 m909 showed much higher (femtomolar) avidity as measured by ELISA, and it bound to FRß positive cells in a dose-dependent manner, but not to parental FRß negative cells. m909 did not compete with folate for the binding to FRß on cells. m909 was not only able to select FRß positive, activated macrophages from synovial fluid cells of arthritis patients as efficiently as folate, but also able to mediate ADCC in FRß positive cells. CONCLUSIONS: Unlike folate-drug conjugates, m909 selectively binds to FRß, does not recognize FRα, and has at least one effector function. m909 alone has potential to eliminate FRß positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. m909 can also be a valuable research reagent.