The proliferative responses of distinct cell subsets from healthy, bacille Calmette–Guérin (BCG)-vaccinated blood donors were assessed after in vitro stimulation with live or UV-killed Mycobacterium tuberculosis and Myco. avium or with soluble extracts obtained from either mycobacterial species. Proliferation of cell subsets was evaluated by flow cytometric determination of 5-bromo-2′-deoxy-uridine incorporation into DNA and simultaneous identification of surface phenotypic markers. In the presence of monocytes, the response to whole (live or killed) bacteria was characterized by a predominant proliferation of CD4+αβ+ T cells and, to a lesser extent, of CD8+αβ+ T cells. Proliferation of CD8+αβ+ T cells was primarily elicited by live rather than killed bacilli (P < 0.05). Conversely, when soluble bacterial extracts were used as stimulators, a preferential proliferation of γδ+ T cells, expressing predominantly Vγ9+ and Vδ2+ T cell receptor chains, was recorded. Moreover, when monocyte-depleted cell populations were directly cultured with live bacteria, a marked proportion of CD3–CD16+ (natural killer (NK)) cells was detected among the responding cells. Although both αβ, γδ and NK cells have been previously shown to react with mycobacteria in vitro, their relative contributions to the response have been difficult to assess. Using a flow cytometric technique which allows direct identification of proliferating cells within complex cell populations, our study demonstrates significant differences in the ability of various mycobacterial antigen preparations to elicit proliferation of distinct cell subsets.