We have previously established that the tissue-specific activity of the hepatitis B virus (HBV) enhancer is mediated by trans-acting cellular factors. Here we have studied in vitro the interactions between the HBV enhancer DNA and cellular factors present in nuclear extracts from both liver and nonliver cell types. The results presented in this study imply the involvement of several distinct, ubiquitous, and liver-specific cellular factors with the HBV enhancer. Sequence analysis of the binding sites for these proteins on HBV DNA showed homologies to sequence motifs known to bind other previously characterized and purified transcription factors including CAAT/enhancer-binding protein. Thus, all of these binding sites may function in concert to activate liver-specific transcription of HBV genes from their respective promoters.