Objective: This study investigated the co-existence of Leishmania chagasi infection andchildhoodleukemia in patients naïve to treatment; this has serious clinical andepidemiological implications.Methods: The seroprevalence of L. chagasi antibodies prior to any treatment was investigatedin children with clinical features of acute leukemia. Serological tests were performed in 470samples drawn from under 14-year-old children from different regions of Brazil with clinicalsuspicion of acute leukemia. Acute leukemia subtypes were characterized by immunophe-notyping using flow cytometry. Morphological analyses of bone marrow aspirates weresystematically performed to visualize blast cells and/or the formation of L. chagasi amastig-otes. Data analysis used a standard univariate procedure and the Pearson’s chi-square test.Results: The plasma of 437 children (93%) displayed antibodies against L. chagasi by indi-rect immunofluorescence assay and enzyme-linked immunosorbent assay tests. Of the 437patients diagnosed from 2002 to 2006, 254 had acute lymphoblastic leukemia, 92 had acutemyeloid leukemia, and 91 did not have acute leukemia. The seroprevalence of L. chagasiantibodies according to the indirect immunofluorescence assay test (22.5%) was similar inchildren with or without acute leukemia (p-value = 0.76). The co-existence of visceral leish-manasis and acute leukemia was confirmed in 24 children. The overall survival of thesechildren was poor with a high death rate during the first year of leukemia treatment.Conclusion: In the differential diagnosis of childhood leukemia, visceral leishmanasis shouldbe considered as a potential concurrent disease in regions where L. chagasi is endemic.