The X gene product of hepatitis B virus (HBV) transactivates a wide variety of promoters, including four promoters on the HBV genome (Rossner, 1992, J. Med. Virol. 36, 101-117). We compared their transactivation efficiencies and investigated whether the spatial organization of the promoters with respect to other cis-acting elements might influence their activities. Eight reporter plasmid constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene were designed such that four had the isolated HBV promoters linked to the CAT gene. In the other four, the CAT gene was inserted downstream to each of the four promoters retained in context in the HBV genome. Cells of the human hepatoblastoma line HepG2 were transfected with each one of these reporters together with an effector plasmid, pRSVX, which allowed expression of X protein. All of these promoters could be stimulated by X protein by approximately 2- to 3.5-fold irrespective of their spatial context in the HBV genome. Mutational analysis of in-frame ATG codons in the X gene provides evidence that transactivator product(s) are produced by internal initiation of translation. Transfection of HepG2 cells with HBV genomes bearing a stop mutation in the X gene at codon 118 resulted in poor production of all viral components. Their syntheses were restored upon transfection of the wild-type X gene.