To investigate whether PreS1 antigen and/or anti-PreS1 antibody in serum might have some significance different from routine hepatitis B virus (HBV) markers in hepatitis B virus infection.Forty-six acute hepatitis B patients and 377 chronic persistent hepatitis patients were examined to assess the clinical significance of serum PreS1/anti-PreS1. HBV family clustering analysis was carried out in a village in Shanghai suburb area, and case-control study on role of PreS1 in HBV spreading was undertaken in Tangshan, Hebei province.PreS1 in HBsAg positive sera from chronic persistent hepatitis patients correlated with HBV DNA (P < 0.05), while the presence of PreS2 in HBsAg positive sera was much more common than that of PreS1 and had no correlation with HBeAg or HBV DNA. Among 46 patients with acute hepatitis B, the appearance of anti-PreS1 antibody in serum correlated with the disappearing or decreasing of both PreS1 and ALT. In antiviral treatment, PreS1 antigen turned negative much earlier than PreS2 and HBsAg in acute hepatitis B patients' sera. Family clustering analysis suggested that people with PreS1 positive in serum were more infective than those with HBsAg positive alone (P < 0.01). The case-control study showed that chronic hepatitis B patients with PreS1 positive in serum played a much more important role in HBV spreading than those with PreS1 negative (P < 0.01) and asymptomatic HBsAg carriers (P < 0.01), and the odds ratios (OR) were 3.8 and 3.2, respectively.Serum PreS1 closely correlates with active virus replication, and PreS1/anti-PreS1 status indicates the outcome of acute hepatitis B. The serum PreS1/anti-PreS1 marker is of some significance in HBV epidemiology as well.