The role of large (pre-S1) and middle (pre-S2) proteins of HBsAg in hepatitis B virus (HBV) infection is not fully known. Therefore, we studied the expression of pre-S proteins in the liver and serum of 26 patients with chronic HBV infection, using immunoperoxidase staining and enzyme immunoassay. Pre-S1 and pre-S2 proteins were detected in a large number of patients in both liver and serum, irrespective of the disease activity. Serial sections showed that most cells positive for HBsAg were also positive for pre-S proteins. The localization of pre-S2 and HBsAg was similar, with cytoplasmic and membranous stainings of hepatocytes, whereas pre-S1 was expressed exclusively in cytoplasm. Serum levels of HBsAg, pre-S1, and pre-S2 of DNA polymerase-positive cases were significantly higher than those of DNA polymerase-negative cases. Membranous display of pre-S2 on hepatocytes was observed more often in DNA polymerase-positive patients, and their serum pre-S2 levels were significantly higher than those without it. The predominant localization of cytoplasmic HBcAg usually was associated with active, ongoing hepatitis. Its expression and DNA polymerase activity were significantly correlated. These results indicate that pre-S proteins in serum and the membranous display of pre-S2 on hepatocytes of patients with chronic HBV infection refect active viral replication, but their expression does not correlate with disease activity.