Abstract Introduction Chagas disease is a parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. Variability in cytokine secretion among chronic Trypanosoma cruzi ‐infected patients might preclude the identification of the pool of antigen specific T cells. The goal of this study was to determine the fraction of T cells responding to T. cruzi antigen measured by the expression of membrane TNF‐α and CD154. Methods A total of 21 chagasic patients, 11 healthy and 5 non‐chagasic cardiomyopathy controls were analyzed. PBMCs were short‐term cultured in the presence of anti‐CD28, anti‐CD49d, anti‐TNF‐α, and TACE (TNF‐α converting enzyme) inhibitor either under T. cruzi‐ lysate or polyclonal stimuli. Cells were stained with anti‐CD3, anti‐CD4, anti‐CD8, and anti‐CD154, and analyzed with flow cytometry. Results CD4+ and CD8+ T cells in chagasic patients displayed higher percentages of membrane‐bound TNF‐α+ and CD154+ compared with controls after T. cruzi ‐antigen stimulation. Both markers displayed a positive correlation in the T cell subpopulations analyzed. Symptomatic chagasic patients were differentiated from asymptomatic patients based on the expression of CD154 and membrane TNF‐α in TCD4+ and TCD8+ compartments, respectively. Conclusions These results show that both markers could be useful for assessing the pool of antigen‐specific T cells in chronic chagasic patients.