CD8+ T lymphocytes are required to protect mice against Mycobacterium tuberculosis, although in early infection the mechanism appears not to be via perforin or granzyme-mediated lysis of the infected target, and may be via interferon-gamma (IFN-gamma) production. We therefore investigated whether CD8+ T cells specific for the immunoprotective 38 000 MW antigen of M. tuberculosis could be detected in infected humans. Using a recombinant vaccinia virus expressing the 38 000 MW antigen of M. tuberculosis (rV38) and a control vaccinia virus (rVras) we demonstrated that both viruses stimulated IFN-gamma production from freshly isolated peripheral blood mononuclear cells (PBMC) in a 36-hr enzyme-linked immunospot assay. Cell depletion and antibody blockade established that the bulk of the 38 000 MW antigen-specific IFN-gamma response was mediated by CD8+, major histocompatibility complex class I-restricted T cells, whereas the anti-vaccinia virus response was predominantly mediated by CD4+ T cells. In further evaluations PBMC from all seven healthy tuberculosis-exposed contacts had a 38 000 MW antigen-specific IFN-gamma response, whereas seven patients with untreated sputum-positive pulmonary tuberculosis had very low levels of 38 000 antigen-specific IFN-gamma-producing cells. These preliminary observations demonstrate the utility of recombinant vaccinia viruses in restimulating freshly isolated CD4+ and CD8+ T cells. The bias towards a higher frequency of IFN-gamma-producing CD8+ T cells in contacts rather than patients may indicate a protective role for CD8+ cells in human tuberculosis.