By transfections of hepatitis B virus (HBV) DNA into five human hepatoma cell lines with the characteristics of differentiated human hepatocytes, three human hepatoma cell lines possessing partial hepatocyte-associated markers, and one non-liver cell line, we demonstrated that the expression of hepatitis B surface and core genes preferentially occurred in hepatoma cell lines with differentiated hepatocyte-associated characteristics. With a heterologous CAT gene as a reporter, the transcriptional activity of the promoter region containing both the distal (SPI) and the proximal (SPII) promoters of hepatitis B surface gene was found to show a preference for differentiated hepatoma cell lines. The SPI promoter which produces a RNA transcript for the synthesis of the large surface protein shows a strong preference, at least 750-fold, for differentiated hepatoma cells, while the SPII promoter which produces RNA transcripts for the synthesis of the middle and major surface proteins shows a moderate preference, about 20- to 59-fold. Further study indicates that this 750-fold preference of the SPI transcriptional activity for differentiated hepatoma cell lines can be attributed to the regulatory sequences of both the SPI and the HBV enhancer regions. These results also imply the important role of the large surface protein of HBV on the hepatocyte-specific infectivity of this virus.