To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.