In order to estimate the magnitude of Leishmania/HIV co-infection, patients with HIV/AIDS at the Brasilia University Hospital, DF, Brazil were used as subjects in a cross-sectional study. One hundred and sixty-three patients were enrolled, seven of whom had visceral leishmaniasis (VL). One hundred and twelve patients (68.7%) were men; 155 (95.1%) had been exposed to HIV infection through unprotected sex. The median age was 37 years (range: 20-74) and the median CD4+ lymphocyte count was 314 cells/microl (range: 2-1600). Symptomatic patients underwent bone marrow evaluations through direct examination of Giemsa-stained films, parasite culture and PCR assay. Blood samples were evaluated by means of an indirect immunofluorescent antibody test (IFAT), an ELISA using a soluble antigen of L. chagasi (ELISA), an ELISA with the rK39 antigen (ELISA-rK39) and a PCR targeted to the kDNA region and to the internal transcribed spacer 1 of the rDNA gene. The proportion of positive results was 2.4% for the IFAT, 12.3% for the ELISA and 4.9% for the rK39 tests. The estimated prevalence was 16%. The PCR in the blood was positive in three patients (1.8%). The prevalence of Leishmania spp. infection is high among HIV patients attending this Brazilian center suggesting that they should be routinely investigated for VL infection.