Despite strong evidence for CD8+ T-cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate. We have evaluated the human macrophage (Mφ) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I-restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules. U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiated U937 cells were capable of efficient phagocytosis of M. tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette–Guérin (BCG) and the virulent M. tuberculosis H37Rv strain. CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M. tuberculosis-specific, HLA class I-matched, purified CD8+ CTL lines and CD8+ T-cell clones. Furthermore, M. tuberculosis-infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mφ. Finally, M. tuberculosis-infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial-specific, HLA-unrestricted γδ+ CTL activity.