MHC class Ia-deficient mice (H2 K b−/− D b−/−) inoculated with the intracellular pathogen Listeria monocytogenes (LM) displayed a three-to fourfold expansion of splenic CD8+ T cells 6 days following infection. Culture of these spleen cells in vitro gave rise to CTL that recognized LM-infected target cells and were restricted by the class Ib molecules, Qa1 b and M3. Exposure of target cells to heat-killed LM (HKLM) rather than live bacteria did not result in CTL-mediated lysis. Target cells pulsed with three LM peptides known to bind M3, f-MIGWII, f-MIVTLF, and f-MIVIL, were recognized by effector cells from both B6 and K b−/− D b−/− animals. In vivo analysis showed that B6 and K b−/− D b−/− mice clear LM from the spleen and liver rapidly with similar kinetics, whereas TAP. 1−/− mice, which are deficient in class Ia and Ib molecules, clear LM slowly upon infection. To establish the in vivo role of CD8+ T cells in K b−/− D b−/− animals, we showed that depletion of such cells from the spleens of immune mice prevented the adoptive transfer of protective immunity to syngeneic recipients. Spleen cells from K b−/− D b−/− mice were also capable of generating responses directed against syngeneic as well as allogeneic class Ia molecules in vitro. Thus, class Ia-deficient animals have a CD8+ T cell repertoire capable of recognizing both class Ia and class Ib molecules and can generate protective immunity to LM.