The aim of this study was to evaluate the prevalence of human immunodeficiency virus (HIV)-Trypanosoma cruzi co-infection in a Buenos Aires health center. A retrospective analysis of the clinical charts of 602 HIV-infected patients was performed. Only 51.3% of the patients were evaluated against T. cruzi. The global co-infection prevalence was 4.2%, being more frequent among injectable drug users (IDU) (8.9% vs. 2.6%, < 0.05). The indication of T. cruzi testing should be stressed for HIV-infected patients, especially in those centers where IDU are assisted.