The analysis of promastigote excreted-secreted antigen (ESA) reactivity with 53 visceral leishmaniasis (VL) cases showed that each sample reacted regardless of the antigen or the Leishmania species used in enzyme-linked immunosorbent assay (ELISA) displayed 100% positivity with the L. (L.) chagasi ESA-blot recognizing bands of molecular weight ranging from 26.5 to 31.5 kDa. The analysis of 160 non-visceral cases showed that 5% of the samples cross-reacted with the L. (L.) chagasi ESA-ELISA and 9.4% reacted with the ESA isolated from L. (L.) amazonensis and L. (V.) braziliensis, whereas a high cross-reaction ranging from 24.4% to 25% was observed with total crude promastigote antigens (PRO-ELISA). The ESA-blot of L. (L.) chagasi tested with non-visceral sera samples showed a cross-reaction with 8.8% of cases; most of these cases represented tegumentary leishmaniasis and only one acute chagasic case. These data lead us to recommend the use of ESA as an alternative antigen in VL diagnosis.