Trypanosoma rangeli infects humans nonpathologically in some areas of Central and South America. Due to morphological and antigenic similarities with T. cruzi, the clear identification of this parasite is an important task. Here, we describe the identification and purification of a specific 48-kDa antigen from T. rangeli. By western blotting analysis, this molecule was not detected in T. cruzi epimastigotes and in Leishmania sp. promastigotes. Fluorescence-activated cell sorter analysis demonstrated that the protein is expressed uniformly by the T. rangeli cell population during axenic culture. Additionally, following immunostaining, a particular subcellular localization (present in organelles) is proposed for this antigen. These results suggest that this 48-kDa antigen may be a useful marker for the identification and characterization of T. rangeli isolates.