To evaluate the usefulness of a real-time PCR for Leishmania DNA in the diagnosis and follow-up of patients with human immunodeficiency virus type 1 (HIV-1) and Leishmania coinfection, Leishmania DNA levels were measured in whole peripheral blood from 25 HIV-infected patients with clinical features suggestive of visceral leishmaniasis. Leishmania DNA was detected in 10 of 25 patients with microscopically confirmed visceral leishmaniasis and in none of those without this disease. Following treatment with liposomal amphotericin B, a clinical response was observed in 9 of 10 patients, in association with significantly decreased parasite loads. Seven patients relapsed clinically a median of 110 days after the end of treatment, in association with substantial increases in Leishmania DNA levels. Leishmania DNA levels correlated with the clinical course of visceral leishmaniasis, and their measurement at diagnosis and during and after treatment seems to be useful in the clinical management of HIV-infected patients with this disease.