Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147–155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147–155. Kdm-expressing target cells saturated with synthetic NPP147–155 (10−5 m) were lysed similarly to Kdw-expressing targets by NPP147–155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm–peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147–155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.