B-cell superantigens (BC-SAgs) are immunoevasins that have evolved in response to innate catalytic IgM antibodies; germ-line encoded immunoglobulins present in the preimmune repertoire independent of prior antigen exposure. Catalysis is the result of a 2-step process that involves first the formation of a non-covalent bond between the BC-SAg and the immunoglobulin followed by covalent bond formation at the catalytic site resulting in target hydrolysis. Tc24 is a recently described Trypanosoma cruzi BC-SAg hypothesized to play a role in evading the humoral response early in the infection period. We previously demonstrated that exposure to Tc24 following immunization or infection resulted in the depletion of the catalytic IgM response, leaving a gap in the catalytic IgM repertoire. The present report compares the BC-SAg properties of wild-type Tc24 (Tc24-WT) to that of 2 recombinant Tc24 isoforms: Tc24-C2 (Cys to Ser mutations in the 2 most-proximal Cys residues) and Tc24-C4 (Cys to Ser mutations in all 4 Cys residues present). BC-SAg activity was assessed by immunizing mice with the respective isoforms and examining the ability of IgM purified from the respective groups to hydrolyze the 3 Tc24 isoforms. In addition, the ability of IgM purified from naive mice to hydrolyze the Tc24 isoforms was also assessed. Immunization with Tc24-WT, Tc24-C2, or Tc24-C4 resulted in loss of IgM-mediated hydrolysis of Tc24-WT. However, the ability of IgM purified from naive mice (previously shown to hydrolyze Tc24-WT) was less effective in hydrolyzing the 2 Tc24 isoforms. These data demonstrate that although the BC-SAg site in the mutants remained intact, their reduced susceptibility to IgM-mediated hydrolysis suggested that structural changes resulting from the Cys to Ser mutations altered accessibility to the catalytic site in the 2 isoforms.