It is widely believed that persistence of live parasites at the primary site of infection is important for maintenance of anti-Leishmania immunity. However, whether this immunity requires only the presence of antigen and not necessarily live replicating parasites has not been investigated. To determine whether non-replicating antigens could induce and maintain anti-Leishmania immunity, we inoculated naïve mice with killed parasites (once or 5 times weekly) either alone or in combination with rIL-12 and challenged them with virulent Leishmania major parasites at different times after inoculation. We found that similar to mice that recovered from virulent live L. major infection, mice inoculated repeatedly with killed parasites were protected against virulent L. major challenge. The protection obtained following 5 weekly inoculations of killed parasites was associated with strong antigen-specific IFN-gamma production by cells from the lymph nodes draining the inoculation site. In contrast, mice that received a single or double inoculation of killed parasites either alone or followed with repeated rIL-12 injection were not protected. Repeated antigen inoculation resulted in increased numbers of the IFN-gamma-secreting CD44(+)CD62L(-) T cells that were comparable in magnitude to that seen in mice with persistent infections. Overall, these results suggest that it is possible to generate and maintain anti-Leishmania immunity for a relatively long period of time in the absence of live replicating parasites. However, a certain threshold of effector cells has to be generated in order to achieve this protection.