Hepatitis B virus (HBV) contains an enhancer element that activates the viral core and X gene promoters. To investigate the transcriptional regulation of the viral S gene promoter, we transfected SK-Hep1 cells with circularized forms of HBV DNAs and their enhancerless mutants. We have found that expression of the S gene, determined by measurement of the appearance of HBsAg in the media and by RNA analysis, is to a large extent enhancer-dependent. This observation was further confirmed by analysis of a series of plasmids containing the chloramphenicol acetyl-transferase (CAT) gene under the control of the S gene promoter and the HBV enhancer element. Interestingly, in contrast to its behavior in SK-Hep1 cells, the S gene promoter is highly active in Alexander cells, in the absence of the enhancer element. This implies that activity of the S gene promoter is cell-type specific.