Hepatitis B virus (HBV) gene expression was investigated in the PLC/PRF/5 human hepatoma cell line and the mouse L-cell line transfected with HBV DNA by modulating the in vitro growth conditions. Both cell lines contain the viral genome integrated into cell chromosomes and both have been shown to produce hepatitis B surface antigen only. Treatment with 5-azacytidine (5-azaC) did not enhance hepatitis B core antigen (HBcAg) synthesis in experimentally transfected mouse L cells. Low levels of hepatitis B e antigen (HBeAg) were detected from the L cells regardless of 5-azaC treatment, and synthesis of HBeAg was dependent on the growth state of the cultures. No HBcAg or HBeAg was detected in PLC/PRF/5 cells with or without 5-azaC treatment. This observation suggests that there is a distinct difference between experimentally transfected cells with HBV and the naturally derived material.