In the present study, the diagnostic value of Trypanosoma cruzi recombinant protein (Tc24) was examined. Although antibodies against Tc24 were detected during natural and experimental T. cruzi infections, specificity studies revealed that sera from T. rangeli-infected mice also recognized to some extent Tc24 protein. In addition, sera from Tc24-immunized mice reacted against a 21 kDa polypeptide in T. rangeli extracts. Detailed analysis of the antibody response against 20-40 peptide localized in the Tc24 amino-terminal domain suggests that this sequence is not expressed by T. rangeli 21 kDa antigen. Therefore, the PCR reaction using oligonucleotides corresponding to a 20-26 peptide clearly demonstrated the specificity of the oligoprobes for T. cruzi identification. Positive signals were also found when using blood samples from T. cruzi-infected mice. Taken together, these results suggest that the PCR-based 20-26 assay may be useful in the specific diagnosis of Chagas' disease.