We previously reported on the isolation of a new rat ATP binding cassette (ABC) transporter, ABCB6. We now report the isolation of the full-length cDNA and genomic clones containing the human ABCB6 gene. ABCB6 is 100% identical to the cloned MTABC3 human ABC transporter and contains the typical ABC signature, Walker A and B motifs. We found that HuABCB6 is expressed at low levels in normal human liver. We found that ABCB6 was overexpressed in human hepatocellular carcinomas compared to paired surrounding non-malignant tissue. We found that there was no difference in ABCB6 gene copy between human liver cancer and its paired non-malignant tissue. Because HuABCB6 was overexpressed in human cancers compared to peri-tumoral tissue in the absence of gene amplification, transcriptional regulation may play an important role in its expression. Therefore, we isolated a 14 kb genomic DNA clone containing the HuABCB6 promoter and 5'-flanking region. The 5'-flanking region contains a CpG island, lacks an appropriately positioned TATA element and contains a number of putative transcription factor binding sites. Two transcription start sites were identified by S1 nuclease mapping at -274 and -296 bp from the start codon. Transient transfection of the HuABCB6 promoter constructs (HuABCB6/1.68, 1.39, 1.13, 0.90, 0.52) containing the luciferase reporter gene resulted in a 1100-2300-fold increase in luciferase activity compared to the empty vector control whereas HuABCB6/1.68 subcloned in the reverse orientation resulted in no activity. We observed a significant decrease in luciferase activity with the promoter constructs, HuABCB6/0.25, 0.15 and 0.06, which indicates that an orientation-dependent functional promoter is contained within our previously predicted promoter region of -315 bp to -565 bp as deletion of this 250 bp sequence resulted in a loss of promoter activity.