The non-classic Human Leucocyte Antigen class Ib molecule, HLA-G, is expressed on the invasive, extra-villous cytotrophoblast in human placenta. HLA-G protects against natural killer (NK)-cell-mediated lysis and may modulate the secretion of cytokines. Aberrant expression of HLA-G has been reported in certain disorders of pregnancy. We have studied the DNA sequences of the putative regulatory region located more than 1.1 kilobases 5' from the start site of transcription (a 244 bp HindIII/EcoRI fragment) of the HLA-G gene and of the promoter region to detect any possible polymorphism. We detected one nucleotide substitution in the HindIII/ EcoRI region and one in the promoter region in the alleles G*01012, G*01013, G*0104, and G*0105N compared to G*01011. Several nucleotide substitutions were detected in the region between the 1.1 kb 5' regulatory region and the promoter region. Alleles can be divided into two groups based on the detected polymorphism. The nucleotide substitutions may have implications for the binding of nuclear factors to the regulatory regions. To our knowledge this is the first study of any polymorphism in the 5'-flanking sequences to the HLA-G gene. Further studies are needed to elucidate the exact impact of the detected polymorphism on levels or developmental regulation of HLA-G expression.