This report shows that lymphoid tissues of mice which have resolved a primary infection with Leisihmania major contain parasite-specific major histocompatibility complex (MHC) class I-restricted cytolytic CD8+ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen-presenting cells pulsed with a cyanogen bromide digest of L. major. In H-2b mice, two distinct populations of CD8+ T cells were identified which both lysed target cells pulsed with L. major-derived peptides but were restricted by a different H-2b class I gene product. Interestingly, these two populations appear to recognize different parasite-derived peptides. It is noteworthy that one Kb-restricted CD8+ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major-derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite-specific MHC class I restricted cytolytic CD8+ T cells for the elimination of L. major by the infected host remains to be determined.