In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.