The hepatitis B virus (HBV) core transcript promoter has been localized to the X gene. At least two other cis-acting elements reside within the X gene. First, enhancer element II partially overlaps the core promoter. Second, a silencer, upstream of the core promoter, negatively regulates the activity of the core promoter. Thus, the organization of cis-acting elements within the X gene is complex. We have used PCR-based deletion mutagenesis to map the HBV (subtype ayw) and woodchuck hepatitis virus (WHV) core promoters. HBV and WHV DNA fragments were cloned into the Xho I and Bgl II-sites of a promoter-less luciferase expression vector pGL2. Transfections were carried out in human (Huh-7) and woodchuck (WLC-3) hepatoma cells. Each construct was co-transfected with a plasmid capable of expression of human growth hormone (HGH) and luciferase activity was normalized to the amount of HGH secreted into the medium. In both cell lines, the HBV core promoter mapped to nucleotides 1348-1650 of the HBV genome and the WHV core promoter mapped to nucleotides 1475-1625 of the WHV genome. We also identified an antisense promoter, between nucleotides 1398 and 1710 of the HBV genome and 1625 and 1825 of the WHV genome. Next, we divided the whole WHV genome into 12 overlapping fragments and cloned them into pGL2 to compare the strength of the core promoter with that of the other WHV promoters and to identify any other region of the WHV genome that might contain antisense promoter activity. Core promoter activity was weaker than the preS1, preS2/S and X promoter activities. Although we found several regions with antisense promoter activity, the fragment between nt 1625 and 1975 consistently had the strongest promoter activity. Finally, the 5 ends of antisense mRNAs coincided with the antisense promoter regions of HBV and WHV using a modified 5 RACE method. Mammalian hepadnaviruses appear to have a bi-directional promoter complex within the X gene.