We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.