Protective immunity against Mycobacterium tuberculosis infection requires the activation of mycobacterium-specific CD8+ T cells, as well as CD4+ T cells. Therefore, optimizing strategies that stimulate CD8+ T cells recognizing dominant mycobacterial antigens, including secreted proteins, may lead to the development of more effective vaccines against tuberculosis. To generate a viral vaccine that is safe in humans, the early secreted protein, MPT64, was expressed in the attenuated vaccinia virus (VV) strain, modified vaccinia virus Ankara (MVA-64). The immunogenicity of MVA-64 was compared with that of the Western Reserve strain of VV (VVWR-64). The replication-defective MVA-64 was as efficient as VVWR-64 in inducing specific antibodies and cytolytic T-cell responses to a defined H-2-Db-restricted epitope on MTP-64. In addition, priming with MPT64-expressing plasmid DNA (DNA-64), and boosting with either MVA-64 or VVWR-64, markedly enhanced MPT64-specific cytolytic and IFN-gamma-producing CD8+ T-cell responses. These findings suggest that MVA may be a suitable vaccine carrier for stimulating mycobacterium-specific CD8+ T-cell responses and may be particularly relevant for developing vaccines for use in regions endemic for tuberculosis and HIV infection.