Chronic Chagas disease occurs in 16 million individuals chronically infected by the protozoan Trypanosoma cruzi in Latin America, and may lead to a dilated cardiomyopathy in 10-30% of patients. A vigorous cellular immune response holds parasitism in check. However, up to now, few T. cruzi proteins have been shown to be recognized by CD8+ T cells from Chagas disease patients. In this study, we designed 94 peptides derived from T. cruzi proteins cruzipain and FL-160, predicted to bind to HLA-A2 molcules. After in vitro binding assays to HLA-A*0201, 26 peptides were selected, and their recognition by PBMC from Chagas disease patients was tested with the IFN-gamma ELISPOT assay. All 26 peptides were recognized by PBMC from at least one patient. Furthermore, a tetrameric HLA-A*0201 complex built with the cruzipain 60-68 peptide that was frequently recognized in the periphery also bound to CD8+ T cells from a heart-infiltrating T cell line obtained from a single patient with Chagas disease cardiomyopathy. Thus, our results suggest that the recognition of CD8+ T cell epitopes in cruzipain and FL-160 may have a pathogenic or protective role in chronic Chagas disease.