Quantification of Leishmania infantum DNA in blood samples by an ultrasensitive quantitative polymerase chain reaction (QPCR) detected parasitemias in different clinical presentations. We observed a large range of parasitemias, more than 9 log values, and could determine the threshold between asymptomatic carriage and disease in the Mediterranean area (approximately one parasite/mL of blood). Based on kinetoplast DNA amplification, this assay had a sensitivity of 0.001 parasite DNA equivalents/mL and detected asymptomatic carriage of Leishmania. It detected parasite DNA in 58% of healthy subjects, while an immunoblot detected specific antibodies in only 16%. For initial diagnosis of disease, this quantitative PCR with blood samples constitutes a non-invasive alternative to bone marrow aspiration. Its main applications are monitoring of drug therapy and follow-up of immunodeficient patients for biologic confirmation of relapses.