Forty-three hybridoma cell lines producing monoclonal antibody to diphtheria toxin were isolated. Based upon their reactivity with various fragments of the toxin and mutant toxin-related proteins, the monoclonal antibodies were subdivided into 10 groups which recognize at least 10 distinct epitopes on the toxin molecule. Specific antibodies directed against both fragments A and B were found to neutralize cytotoxicity, both in vitro and in vivo. Neutralization was found to correlate with antibody-mediated inhibition of toxin binding to its eukaryotic cell surface receptor. The results presented suggest that the Pro378 and/or Gly431 of mature toxin are part of, or close to, the toxin receptor-binding domain. In addition, antigenic determinants in the C-terminal portion of fragment A, as well as a portion of the toxin defined by the tox-3 and tox-45 nonsense mutations (i.e. ca 31,000-42,000 daltons) appear to be juxtaposed to the receptor-binding domain and may form a secondary binding region.