We previously demonstrated that Bacillus Calmette-Guerin (BCG) immunization activated D(b) restricted CD8+ cytolytic T lymphocyte (CTL) recognizing target cells incubated with mycobacterial culture filtrate. Here, we show that in vitro restimulation of spleen cells from BCG vaccinated or Mycobacterium tuberculosis infected mice with culture filtrate antigens leads to the appearance of a high percentage of D(b) restricted IFNgamma synthesizing CD8+ T cell blasts. Transporter associated protein-2 mutated RMA-S cells incubated with soluble culture filtrate proteins had their MHC class I D(b) but not K(b) molecules stabilized at the surface indicating that only D(b) ligands might be generated by antigen presenting cells. MHC class I bound peptides were acid eluted from the surface of RMA-S cells incubated with M. tuberculosis culture filtrate proteins. The crude peptide preparation was able to sensitize RMA-S cells for recognition by culture filtrate-specific cytolytic T cells. Peptides were subsequently fractionnated by reverse-phase high performance liquid chromatography and the main biological activity was identified in two fractions. These results provide a further evidence that the processing of exogenous culture filtrate proteins in vitro leads to the presentation of a restricted number or even a single immunodominant peptide to culture filtrate-specific CD8+ T cells.