ABSTRACT More than 80% of mammalian antibody-secreting cells reside in the intestinal mucosa and secrete antibodies of the immunoglobulin A (IgA) isotype. IgA deficiency is common in humans and predisposes toward various intestinal pathologies including inflammatory bowel disease, celiac disease, and allergy. However, despite decades of research, the specificity of IgA remains elusive. IgA coats a fraction of the intestinal microbiota, yet the identities of the bacteria targeted by IgA and the types of humoral responses involved remain unknown. IgA may also target dietary antigens, but it is unclear whether most IgA is directed against dietary antigens, commensal microbiota, or other intestinal antigens. Lastly, specific antigens recognized by homeostatic IgA antibodies have not been described. Here we present extensive preliminary data characterizing the commensal bacteria targeted by IgA using a novel method involving bacterial flow cytometry and 16S rRNA gene sequencing, termed IgA-Seq. We further present a method for cloning and expression of recombinant monoclonal antibodies from single intestinal IgA-producing cells and rapid characterization of their reactivity against microbiota using IgA-Seq. Finally, we detail a plan for determining the antigenic targets of these antibodies with the following specific aims: 1) To characterize the origin and frequency of commensal-specific B cells; and 2) To determine the specificities of single IgA antibodies. These data will be integrated to develop a working understanding of the commensal bacteria and specific bacterial and/or dietary antigens that drive homeostatic IgA responses. An understanding of the specificity of IgA will clarify hypotheses regarding its elusive function. Further, manipulation of the IgA response may represent a promising therapeutic approach to various microbiota-associated pathologies including inflammatory bowel disease, celiac disease, obesity, and diabetes, but cannot be achieved without a thorough understanding of its specificity. Lastly, our studies will generate a large panel of monoclonal antibodies specific for commensal microbiota that may allow manipulation and characterization of defined bacterial subsets in healthy or dysbiotic microbiota and may have applications as research, diagnostic, or therapeutic tools.