Flow cytometry has proven to be a useful tool for the investigation of cytokine synthesis by selected cell subpopulations. While most reports have used mitogen stimulation or long-term cultures with antigen, we describe here a novel method to allow the detection of rare mycobacterial antigen-specific cytokine synthesizing cells within one day. The most important feature of this method is the use of an FITC-conjugated isotype-matched control antibody to identify and exclude cells which fluoresce non-specifically. With this technique, we demonstrate interferon-γ ( IFN-γ) staining in 785 cells per 1×105 T cells counted, in mycobacterial antigen-stimulated peripheral blood mononuclear cells from a BCG-vaccinated subject. In comparison, only 14 IFN-γ-staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1×105 T cells are stained by an irrelevant control antibody. Specific responses are detectable after 12 h of in vitro culture, and peak at 24 h. In volunteer health care workers, IFN-γ staining correlated with IFN-γ production using a published ELISPOT assay (r=0.927). IFN-γ staining was also higher in PBMC from mantoux skin test-positive volunteers, compared to cells from skin test-negative subjects (p=0.0045). Flow cytometry following short-term culture can thus be used for enumeration of antigen-specific IFN-γ synthesizing cells.