The recombinant proteins KMPII, TRYP, and LACK of Leishmania infantum were produced in baculovirus-infected Trichoplusia ni larvae and used to analyze the seroreactivity of 165 dog serum samples by the multiple-well ELISA technique (57 infected dogs with clinical signs, 46 naturally infected and 11 experimentally infected; and 108 non-infected dogs, 76 from non-endemic areas and 32 from endemic areas). Recombinant (r) KMPII was the most recognized antigen, as the majority of infected dogs seroreacted against it (0.75). This is the first report of seroreactivity against rTRYP (0.51) and rLACK (0.42) in L. infantum-infected dogs, since previous studies using recombinant TRYP and LACK proteins produced in prokaryotic systems failed to detect specific seroreactivity. All non-infected dogs were negative for rTRYP and rLACK, and only one of the 32 from endemic areas seroreacted against rKMPII. The results demonstrate that L. infantum-infected dogs develop humoral immunity against rKMPII, rTRYP, and rLACK antigens. There was substantial agreement between crude total L. infantum antigen (CTLA)-based ELISA and rKMPII ELISA (kappa=0.664), although this was higher than that found between the CTLA-based ELISA and rTRYP (kappa=0.427) or rLACK (kappa=0.343) ELISA, which can be interpreted as fair and moderate agreement, respectively. Ninety-three percent of the infected dogs analyzed developed specific antibodies against at least one of these three recombinant antigens. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement (kappa=0.880) with CTLA-based ELISA was observed, with a specificity of 0.97 and a sensitivity of 0.93 in relation to CTLA-based ELISA. Further studies using purified recombinant antigens in a single-well test or individually, depending on the objective of the study, are warranted.