Individuals with frequent exposures to agents spread through the parenteral route show a high prevalence of Leishmania seropositivity in Spain. However, the frequency of positive polymerase chain reaction (PCR) results for Leishmania in blood in this setting remains unknown. In this study, L. infantum kinetoplast DNA (kDNA) was found in blood from 23 (24%) of 95 asymptomatic individuals with a serum Leishmania antibody titer > or = 1:20 and in none of 44 seronegative individuals. The greater the antibody titer, the greater the proportion of PCR-positive samples, but 16 (20%) of 81 individuals with antibody titers < or = 1:40 tested positive by PCR. Nine (37%) PCR-positive and 22 (19%) (P = 0.03) PCR-negative individuals showed a positive leishmanin skin test result. This results show that a remarkable proportion of asymptomatic Leishmania-seropositive individuals at risk for parenterally transmitted infections carry Leishmania kDNA in blood. This is more common in subjects with a high serum Leishmania antibody level and a positive leishmanin skin test result.