The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa. Based on these similarities, we have designated this gene family tolT. Immunization of mice with recombinant TolT generates a population of CD4⁺ T lymphocytes that recognize T. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN- and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected with T. cruzi also produce significant levels of IFN- when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T. cruzi.