Major histocompatibility complex (MHC, HLA in humans) class I molecules play an important role in cellular immunology by presenting viral, tumor-associated or minor histocompatibility antigen-derived peptides to T cells. Tumor cells frequently fail to express one or more of the different MHC class I loci (HLA-A, -B and -C), thereby avoiding elimination by T cells. In primary human melanomas as well as melanoma cell lines, HLA class I expression is frequently down-regulated in a B locus-specific manner. The HLA class I promoter contains a number of cis-regulatory elements located upstream of the transcription-initiation site, among them enhancer A and an interferon-stimulated response element. In the present study, we show that novel sequences located 13 to 33 bp downstream of the transcription-initiation site mediate HLA-B locus-specific down-regulation in human melanoma cell lines. Furthermore, involvement of the +13 to +33-bp region in HLA-B locus-specific down-regulation in vivo is supported by in vitro experiments showing locus-specific binding of protein complexes to the +13 to +33-bp region.