Twenty monoclonal antibodies against human erythrocyte spectrin were generated using purified native spectrin as the immunogen. Thirteen out of 20 monoclonal antibodies reacted to the alpha subunit, and the remaining seven monoclonal antibodies reacted to the beta subunit in Western blot analysis. One anti-beta subunit and two anti-alpha subunit monoclonal antibodies cross-reacted with the cell lysate of the non-erythroid T. lymphoma, J. Jhan, cell line. Identification of 20 monoclonal antibodies to the respective tryptic domains was also achieved. The specificity of these antibodies to respective tryptic domains of spectrin confirms the alignments and uniqueness of each of the previously identified five domains in the alpha subunit and four domains in the beta subunit, and establishes the identity of proteolytic daughter peptide fragments.