Previously, we described H-2K bW9 (K bW9), an engineered variant of the murine MHC class I molecule H-2K b (K b), devoid of the central anchor (“C”) pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of K b and K bW9 are largely nonoverlapping in vivo. On the basis of these observations, we used K bW9 and K b to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of K bW9 and K b. Six of six K b-specific mAbs, directed against different parts of the molecule, recognized K bW9 well, albeit at different levels than K b. Furthermore, K bW9 readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, K bW9 mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between K b and K bW9. Interestingly, when K bW9 was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against K b and was specific for peptide-independent MHC epitopes. By contrast, K b-induced alloreactive CTLs recognized K b in a peptide-specific manner, did not cross-react on K bW9, and were present at much higher frequencies than those induced by K bW9. Thus, induction of rare peptide-independent CTLs depended on unique structural features of K bW9, likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.