Transcription of major histocompatibility complex class I genes is controlled by the class I regulatory complex in the 5' flanking region. To investigate the molecular basis of this region, we studied the polymorphism of the promoter of the HLA-B locus extending from the ATG transcription initiation signal to -284 base pairs (bp) which includes a number of cis-acting elements: interferon response sequence (IRS), enhancer A and enhancer B. Genomic DNA from 35 homozygous cell lines from the 10th International Histocompatibility Workshop and from eight heterozygous panel members was amplified using two primers designed to specifically amplify the HLA-B locus. The double-stranded polymerase chain reaction products were sequenced using the cycle sequencing technique and an ABI 373A automatic sequencer. Promoter sequences of thirty-one different HLA-B alleles were determined in this study. Within the 284 bp upstream of the ATG signal, base substitutions were observed in 23 different nucleotide positions. Our study shows a high degree of polymorphism of the HLA-B promoter region, but conserved sequences of the known cis-acting elements with the exception of enhancer B in which there are two base substitutions for B7 and B42 (position -93 and position -95). The 23 polymorphic sites can be grouped into 12 different HLA-B promoter types (groups A to M) for 31 HLA-B locus alleles. Some of the groups of alleles sharing the same promoter sequence such as, for example, group A with B51, B52, B53, and B35, might have been predicted on the basis of serological similarity and/or exon 2, 3 sequence. In other groups, such as G (B18, B37, B27), it could not have been anticipated from serological experience that B18 and B27 carry the same promoter. Several sequencing errors were detected in the HLA-B promoter sequences published previously.