Chronic human Chagas' disease ranges from an asymptomatic to a severe cardiac clinical form. The involvement of the host's immune response in the development and maintenance of chagasic pathology has been demonstrated by several groups. We have shown that activated T-cells lacking CD28 expression are increased in the peripheral blood of chagasic patients (CP), suggesting a relationship between these cells and disease. In order to better characterize this cell population, determining their possible role in immunoregulation of human Chagas' disease, we evaluated the expression of TCR-Vbeta regions 2, 3·1, 5, 8 and 17, as well as the expression of IFN-γ, TNF-α, IL-4 and IL-10 by CD28+ and CD28− cells from polarized indeterminate and cardiac CP. Flow cytometric analysis demonstrated equivalent TCR-Vbeta usage between CD4+CD28+ and CD4+CD28− cells from all groups (chagasic and healthy controls). However, there was a predominance of Vbeta5 expression in the CD28+ and CD28− populations in the CP groups (indeterminate and cardiac). Interestingly, CD8+CD28− cells from CP, but not from nonchagasic individuals, displayed a reduced frequency of most analysed Vbetas when compared with the CD8+CD28+ subpopulation. Comparison of V-beta expression in CD28+ or CD28− cell populations among individuals from different groups also showed several interesting differences. Functionally, cardiac CP displayed a higher frequency of IFN-γ, TNF-α and IL-4 producing lymphocytes than indeterminate CP. Correlation analysis between the frequency of cytokine expressing cells, and the frequency of CD4+ T-cells with differential expression of CD28 demonstrated that CD4+CD28− T-cells were positively correlated with TNF-α in cardiac and with IL-10 in indeterminate CP, suggesting that these cells might have an important regulatory role in human Chagas' disease.