Transcription from the hepatitis B virus (HBV) nucleocapsid promoter is regulated in a cell-type-specific manner and can be modulated by the HBV enhancer I element. Mutagenesis analysis of the nucleocapsid promoter demonstrated that the two Sp1-binding sites (CpB and CpC) in the minimal promoter were the major determinants of transcriptional activity in the dedifferentiated hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3. In contrast, binding sites for transcription factors located immediately upstream (CpE) and downstream (CpF) of the two Sp1-binding sites were shown to be important determinants of nucleocapsid promoter activity in the differentiated hepatoma cell lines, Huh7 and HepG2. The role of these elements in the regulation of the nucleocapsid promoter activity correlated with the formation of specific DNA-protein complexes between Huh7 and HepG2 nuclear extracts and the CpE and CpF region sequences. Characterization of the influence of the nucleocapsid promoter mutations in the presence or absence of the enhancer I demonstrated that modification of individual transcription factor binding sites does not prevent enhancer-mediated activation of transcription from the nucleocapsid promoter. These results indicate that differentiated-hepatoma-specific transcription factors plus the Sp1 transcription factor interacting with the nucleocapsid promoter and the enhancer I regulatory region contribute to the level of transcription from this HBV promoter.