Class I molecules of the MHC bind foreign and endogenous peptides allowing recognition by the TCR on CTL. The recognition and killing of cells infected with lymphocytic choriomeningitis virus (LCMV) depends on the recognition of LCMV peptides bound to class I MHC. Mutations in class I MHC molecules have enabled the delineation of regions in the class I molecule important for binding peptides and for interaction with the TCR. We have constructed a library of class I mutants using saturation mutagenesis and report a phenotypic change resulting from a single amino acid substitution that results in the heteroclitic (increased) killing of LCMV-infected cells. This amino acid change, asparagine to serine at position 30, is in a conserved region of the class I molecule contacting the alpha 3 domain. This mutation does not result in increased expression of the class I molecule on the cell surface, does not affect the binding of CD8, and does not affect allogeneic recognition. Cold target experiments show that this heteroclitic killing is due to increased recognition by CTL. These data point toward a critical function for this region of the class I molecule in the binding of peptides or their presentation to CTL.