Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.