Human tonsillar lymphocytes immunized in vitro with diphtheria toxoid were fused with the heteromyeloma cell line SP2/HPT. Hybrids were selected on hypoxanthine-thymidine aminopterin (HAT) medium and assayed for antibody production both by ELISA and Western blotting. Four stable hybridoma cell lines producing 3-7 micrograms/mL of human IgG1 antibodies were isolated. In Western blotting studies these antibodies show binding to the B fragment of diphtheria toxin. Cells from the subclone P2-13/G35 were injected i.p. in pristane-primed nude mice, and ascites fluid was collected after 4-6 weeks. The monoclonal antibody neutralized the cytotoxicity of diphtheria toxin in the Vero cell assay system. This human monoclonal antibody may have a potential in the immunotherapy of diphtheric disease.