Clearance of the intracellular bacterial pathogen Listeria monocytogenes requires antigen-specific CD8(+) T cells. Recently it was shown that activation of class Ib major histocompatibility complex (MHC)-restricted CD8(+) T cells alone is sufficient for immune protection against listeriae. A major component of the class Ib MHC-restricted T-cell response is T cells that recognize formylated peptide antigens presented by M3 molecules. Although three N-formylated peptides derived from L. monocytogenes are known to bind to M3 molecules, fMIGWII is the immunodominant epitope presented by M3 during infection of mice. The source of fMIGWII peptide is the L. monocytogenes lemA gene, which encodes a 30-kDa protein of unknown function. In this report, we describe the generation of two L. monocytogenes lemA deletion mutants. We show that lemA is not required for growth of listeriae in tissue culture cells or for virulence during infection of mice. Surprisingly, we found that fMIGWII-specific T cells were still primed following infection with lemA mutant listeriae, suggesting that L. monocytogenes contains at least one additional antigen that is cross-reactive with the fMIGWII epitope. This cross-reactive antigen appears to be a small protease-resistant molecule that is secreted by L. monocytogenes.