Hepatocyte nuclear factors 4 alpha (HNF4alpha) and 3 beta (HNF3beta) are members of a group of liver-enriched transcription factors (LETFs) that play important roles in regulating the replication of hepatitis B virus (HBV). Using cell culture and animal models, we showed that HNF4alpha supports HBV replication in nonhepatic cells and HNF3beta inhibits HBV replication. However, the expression of HNF4alpha and HNF3beta in the liver tissue of chronic HBV-infected patients and the relationship between the levels of HNF4alpha and HNF3beta and HBV replication are unclear. In this study, liver biopsy specimens from 86 chronic HBV-infected patients were collected. The expression levels of HNF4alpha, HNF3beta, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) were detected by an immunohistochemical technique and the level of HBV DNA was checked by in situ hybridization with serial sections from liver biopsy tissue samples. We show here that samples with higher levels of HNF4alpha expression also have higher levels of HBsAg, HBcAg and HBV DNA. In contrast, in samples with higher levels of HNF3beta expression, levels of HBsAg, HBcAg and HBV DNA were lower. There was a positive correlation between HNF4alpha expression and HBV replication, and a negative correlation between HNF3beta expression and HBV replication, in the liver of chronic HBV-infected patients. This suggests that HNF4alpha and HNF3beta likely participate in HBV replication in patients with HBV infection, or that HBV replication may somehow influence the expression of HNF4alpha and HNF3beta in the liver.