A2 proteins are expressed in the amastigote stage of Leishmania donovani and are composed predominantely by a conserved repetitive element. Here, we have investigated the presence of anti-A2 antibodies in a panel of American Visceral Leishmaniasis (VL) sera. Anti-A2 antibodies were detected by ELISA, using a recombinant A2 protein containing a tag of six histidine residues (A2-HIS), in 77% of patients sera with symptomatic VL and in 87% of sera from dogs that tested positive in Leishmania immunofluorescent-antibody test (IFAT) or in the parasitological evaluation. Anti-A2 antibodies were also detected in 14 out of 15 symptomatic and in 10 out of 13 asymptomatic dogs. In addition, among the asymptomatic/anti-A2 positive animals, 9 were also positive for the presence of parasites. No significant cross reactivity was observed with sera of animals with other common canine diseases. Our findings suggest that A2 protein is a potential tool for the diagnosis of VL in the New World, and will be particularly useful for diagnosis of dogs.