Early studies of influenza virus-specific CD8+ T cell-mediated immunity indicated that the level of CTL activity associated with H2D b is greatly diminished in mice that also express H2K k. Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2K k on the H2D b-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-γ-based flow cytometric assay. The depressive effect of H2K k was very apparent for the influenza D b PA 224 epitope and was also reproduced when CTL activity was measured for H2D b-expressing targets pulsed with the immunodominant NP 366 peptide. The secondary CD8+ IFN-γ+ D b NP 366-specific response was much greater in parental H2 b than in H2 k× b F 1 mice, but the sizes of the CD8+ sets specific for K k NP 50 and D b NP 366 were essentially equivalent in the F 1 animals. Thus, although the immunodominance profile associated with D b NP 366 is lost when H2K k is also present, the response is still substantial. A further, MHC-related effect was also identified for the K k NS1 152 epitope, which was consistently associated with a greater CD8+ IFN-γ+ response in H2K k D b recombinant than in (H2K k D k× H2K b D b) F 1 mice. The diminished D b PA 224 response in H2 k× b F 1 mice was characterized by loss of a prominent Vβ7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies.