The heart is the most commonly affected organ in chronic Chagas' disease, and lymphocytic myocarditis is often observed. However, the pathogenesis of chronic Chagas' heart disease is controversial. The purpose of this study was to determine whether in vivo T. cruzi antigens could be detected in hearts from patients with chronic Chagas' disease and to investigate whether a correlation between these antigens and the intensity of myocardial inflammation exists. We studied 16 patients with chronic Chagas' heart disease. Ten patients had severely impaired left ventricular function and refractory heart failure, and six had episodes of sustained ventricular tachycardia without severe left ventricular dysfunction. Eight patients underwent magnetic resonance imaging with gadolinium enhancement to guide endomyocardial and surgical biopsies to sites with more intense inflammatory processes. Myocardial specimens were studied with immunohistochemical techniques by using rabbit anti-T. cruzi immune serum to detect the presence of T. cruzi antigen. All patients had evidence of some myocarditis in at least one myocardial fragment. T. cruzi antigen was detected in 11 (69%) patients. T. cruzi antigens were detected in 10 (71%) of 14 regions with histopathologic evidence of moderate or severe myocarditis. In contrast, T. cruzi antigens were detected in 3 of 18 regions with only mild or absent myocarditis. There was a statistically significant correlation between the presence of T. cruzi antigens and moderate or severe myocarditis (chi-square = 5.169, p = 0.023). The results of this in vivo study demonstrate that T. cruzi antigens are frequently detected in chronic Chagas' heart disease. In addition, there is an association between the intensity of the inflammatory process and the presence of T. cruzi antigens. The presence of the T. cruzi antigen and its correlation with the severity of myocardial inflammatory process provide strong supportive evidence for the role of T. cruzi even in the chronic forms of Chagas' heart disease.