Antibodies against the Trypanosoma cruzi ribosomal P2 protein (TcP2) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2 molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2s. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2s associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2 (GST-TcP2 or Hist-TcP2) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2 were also able to elicit antibodies against the TcP2 C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2 C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the 1-adrenergic receptor. Thus, antibodies against the TcP2 C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.