Hepatitis B virus (HBV) enhancer is a cis-acting element capable of potentiating transcription with liver cell specificity. To assess the regulatory function of the enhancer during viral gene expression, we employed whole genome transfections in both liver- and non-liver-derived cell lines and compared them with those of enhancerless genome transfections. Using a transient scheme of expression, the effect of enhancer deletion on the transcription of HBV genes was investigated. S1 nuclease analysis showed that deletion of the enhancer sequences did not alter the specificity of the HBV transcripts. Also, the nuclear run-on analysis demonstrated that the influence of enhancer on the HBV gene expression is at the transcriptional level. We further noted that in the absence of the enhancer, synthesis of the pregenomic RNA which originates from the core/pregenomic promoter was markedly affected. This result then implicates an additional role of the enhancer during the replication of HBV.