The serodiagnostic potential of recombinant ORFF protein (rORFF) from Leishmania infantum was assessed by ELISA. Of 49 sera from confirmed cases of visceral leishmaniasis (VL), all were seropositive using 5 ng of rORFF and serum diluted 1:20, while only 38 were positive with 500 ng of soluble antigen (SA) and 44 were positive by a direct agglutination test. There was also a positive correlation between spleen size and level of seropositivity with rORFF or SA. The reciprocal endpoint titer with rORFF was 1,280 for sera from VL patients, but < 20 with sera from malaria, filariasis, and tuberculosis patients, as well as with sera from healthy individuals from endemic and non-endemic areas. Sera from 10 confirmed cutaneous leishmaniasis cases from Turkey were negative or only weakly positive with rORFF although 9 were positive with SA. Thus, rORFF protein appears useful as a sensitive reagent for the differential diagnosis of VL caused by the Leishmania donovani complex.