Worldwide, tuberculosis (TB) is the most frequent coinfection with human immunodeficiency virus-1 (HIV-1), and active TB enhances the progression of HIV-1 disease in dually infected subjects. In the microenvironment of Mycobacterium tuberculosis (MTB)-infected foci, where HIV-1-infected CD4 T-cells come into contact with MTB-infected macrophages, the direct interaction of the two cell types in the activation of latent HIV-1 may be important. In this study we sought to determine whether MTB-infected human primary mononuclear phagocytes-namely, alveolar macrophages (AMs) and their less mature blood precursors, monocytes-activate HIV-1 in a T-cell line stably transfected with an HIV-1 long terminal repeat (LTR) reporter construct (1G5 cells) and induce HIV-1 expression in T-cells from HIV-1-infected subjects. MTB-infected monocytes and AMs, and not Mycobacterium avium-infected cells, activated HIV-1 LTR in 1G5 cells in the presence and absence of HIV-1 tat. Transactivation of HIV-1 LTR by MTB-infected mononuclear phagocytes was mediated mainly by tumor necrosis factor-alpha. In AMs, but not monocytes, membrane tumor necrosis factor-alpha contributed to transactivation of HIV-1 LTR. MTB-infected MNs from 60% of HIV-infected subjects induced HIV-1 LTR in 1G5 cells as well. Furthermore, HIV-1 transcription was induced in autologous T-cells from 30% of the HIV-1-infected subjects. We therefore conclude that MTB-infected mononuclear phagocytes can transactivate HIV-1 in CD4 cells. Transactivation of latent HIV-1 in CD4 T-cells by MTB-infected mononuclear phagocytes may in part be responsible for increased HIV activity at sites of MTB infection during dual infection in vivo.