Sera from 38 Paraguayans with positive skin tests to Leishmania braziliensis panamensis and 51 sera from Paraguayan patients in different stages of Chagas' disease were analyzed by Western blotting using antigen from Trypanosoma cruzi Y strain epimastigotes and from L. b. panamensis promastigotes. Using a single serum sample, distinctive patterns of IgG antibody response to both antigens were identified allowing the differentiation between T. cruzi infection, Leishmania infection, and probable double infection. Sera from patients with T. cruzi infections consistently recognized bands of ∼25 kDa, 38 kDa, and >97 kDa in T. cruzi antigen lysates and recognized a band of 38 kDa in Leishmania lysates. Sera from patients with Leishmania infections recognized a band of 66 kDa in Leishmania antigen lysates but did not consistently recognize any bands using T. cruzi antigen. Sera from patients with probable double infections recognized all bands normally detected by individual sera from patients infected with either T. cruzi or Leishmania. In our study population, T. cruzi infection among leishmaniasis patients was as frequent as among individuals free of leishmaniasis.