We report the identification of a subfraction isolated by gel filtration liquid chromatography from a non-protective Leishmania infantum extract, which protects Balb/c mice against Leishmania major infection, in conjunction with a bacterial adjuvant. Protected mice exhibited cell-mediated immunity, as assessed by increased numbers of CD4+ and CD8+ spleen cells, and the production of IFN gamma, as compared to mice who received only the adjuvant before infection.