The occurrence of leishmaniasis patients carrying a double infection with Trypanosoma cruzi has been suspected but not proved. In this study, we analyzed sera of leishmaniasis patients from a region endemic for both parasites by using immunoblotting with epimastigotes and a purified antigen specific for T. cruzi (Ag 163B6). Seven of 12 patients showed a pattern of bands characteristic of chagasic patients reacting with antigens with molecular weights of 131, 125, 116, 111, 51–45, and 43 kD, and positive reactivity with Ag 163B6, Xenodiagnosis for T. cruzi was carried out in all patients; this technique has a positivity rate of 50% in chronic chagasic patients. The presence of T. cruzi trypomastigotes was shown in the blood of three, thus confirming the existence of a double infection in humans. Since the two parasites possess cross-reacting antigens, it may be assumed that previous infection with one of the parasites may affect the course of subsequent infection with the other. Nevertheless, T. cruzi infection did not prevent the appearance of typical leishmaniasis lesions. Therefore, antigenic cross-reactivity is unable to induce a sterilizing immune response against Leishmania.