A new recombinant K39 immunochromatographic test (ICT) was compared with the immunofluorescent antibody assay (IFA) for the rapid serological diagnosis of visceral leishmaniasis (VL) in Apulia, Southern Italy. A total of 264 individuals were tested, including 19 patients with VL (three of which were HIV positive), 67 individuals with suspected VL, 40 healthy controls and 138 patients with other diseases. The ICT was positive in all 19 patients with VL and negative in sera from the remaining individuals. Both the sensitivity and specificity of ICT was 100%. The ICT also worked well in HIV-Leishmania co-infected patients. Antibodies to Leishmania detected by the IFA and ICT remained at detectable levels for up to 12-24 months. A positive reaction by the ICT was detectable at a serum dilution of up to 1:20,480, indicating that a strong immunoresponse is mounted against the recombinant K39 antigen. In conclusion, the ICT is highly sensitive, specific, rapid, noninvasive and cost effective (euro8.43 for ICT and euro12 for IFA) in the diagnosis of VL in areas of low VL endemicity.