The expression of MHC class I genes has been thought to be regulated by two major cis-acting regulatory elements. The first region, enhancer A (Enh A) spanning from positions -210 to -165 contains perfect palindrome (PP), TGGGGATTCCCCA. The PP is well-conserved both in mouse and human MHC class I genes, even though the PP is disrupted by 2 bp substitutions (TGAGGATTCTCCA) in HLA-C genes. Three proteins binding to the Enh A of HLA-A and -B locus genes, but very weakly or nearly not to the Enh A of HLA-C locus gene have been identified. To determine functional importance of the PP for binding of trans-acting protein, mutant DNA probes were made by site-directed in vitro mutagenesis and then electrophoretic mobility shift assay was performed. HLA-A mutant DNA probe, in which the PP is disrupted, shows the same nuclear protein binding pattern as that of the HLA-C gene, and HLA-C mutant DNA probe, in which the PP is introduced, shows the same nuclear protein binding pattern as that of the wild type HLA-A gene. These data suggest that the perfect palindrome and its cognate DNA binding nuclear protein play an important role in the HLA class I gene regulation, and thus the lower expression of HLA-C antigen may be ascribed to no or very weak factor binding to the nonpalindromic sequences of HLA-C upstream DNA.