Natural killer (NK) cells have been shown to express a clonally distributed ability to recognize HLA class I alleles. The previously defined NK clones belonging to "group 1" recognize HLA-C*0401 (Cw4) and other HLA-C alleles sharing Asn at position 77 and Lys at position 80. Conversely, the "group 2" NK clones recognize HLA-Cw*0302 (Cw3) and other HLA-C alleles characterized by Ser at position 77 and Asn at position 80. We assessed directly the involvement of these two residues in the capacity of NK cell clones to discriminate between the two groups of HLA-C alleles. To this end, Cw3 and Cw4 alleles were subjected to site-directed mutagenesis. Substitution of the amino acids typical of the Cw3 allele (Ser-77 and Asn-80) with those present in Cw4 (Asn-77 and Lys-80) resulted in a Cw3 mutant that was no longer recognized by group 2 NK cell clones, but that was recognized by group 1 clones. Analysis of Cw3 or Cw4 molecules containing single amino acid substitutions indicates roles for Lys-80 in recognition mediated by group 1 clones and for Ser-77 in recognition mediated by group 2 clones. These results demonstrate that NK-mediated specific recognition of HLA-C allotypes is affected by single natural amino acid substitutions at positions 77 and 80 of the heavy chain.