Monoclonal antibodies produced against T. cruzi microsomal fraction (Mc) were used to investigate the presence of molecular mimicry between the parasite and mammalian tissues. A total of 42 cell lines secreting anti-Mc antibodies were characterized and selected by ELISA, dot blotting and Western blotting assays. Twenty seven supernatants reactive with Mc and/or parasite cytosol (CS) also reacted with human myocardial and/or skeletal muscle antigens by dot blotting assay. Twelve among those cross-reactive hybridomes, which happen to be all of the IgM isotype and to recognize structures on the surface and/or flagellum of the parasite, were selected for cell cloning. Western blotting analysis of these 12 monoclonal antibodies revealed that they mainly recognized bands of 65, 45, 34 and 27 kDa on myocardium and bands of 71, 59, 44 and 30-27 kDa on skeletal muscle. Moreover, seven among them, when assayed by immuno-histochemistry on human and hamster myocardium and skeletal muscle, recognized cytoplasmic antigens, although the monoclonal antibodies 5F2 and 5A9B11 did also bind to the vessel muscle layer. Competitive assays proved the specificity of tissue structures recognition by these monoclonal antibodies. Moreover, this reactivity resulted to be organ specific as they failed to react on lung, stomach and kidney samples. These results demonstrate the cross-reactivity of mammalian and parasite antigens, thus supporting the possibility that molecular mimicry plays a central role in the development of chagasic cardiomyopathy.