There are few studies on cell‐mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using 51Cr‐labelled K562 cells as targets. Results are reported as lytic units (LU=number of cells required for 30% lysis) per million PBMC. Exposure of patients’ PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8±5.0 LU/106 PBMC; mean±s.d. than indeterminate (11.5±3.6 LU/106) and symptomatic cardiomyopathy (7.8±2.5 LU/106). Normal control PBMC stimulated with T. cruzi antigen had 4.36±1.31 LU/106 PBMC against K562. Addition of recombinant interferon‐gamma (IFN‐γ) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL‐12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL‐12 and a neutralizing anti‐IFN‐γ antibody did not change IL‐12‐induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up‐regulated by IL‐12.