The objective of this work was to evaluate whether postnatal hepatitis B immunization failure in children is caused by prenatal infections. A prospective study was conducted from October 2006 to September 2008. Fetal samples from HBsAg-positive mothers were retrieved by either amniocentesis or cordocentesis (percutaneous umbilical blood sampling [PUBS]). Hepatitis B virus (HBV) serologic markers (HBVM) and quantitative HBV DNA assays were performed to assess prenatal infection. All neonates were given combined HBV immunoprophylaxis after delivery. The newborns were followed up with HBV serologic testing at 1 year old. For the 252 pregnant women recruited, 16 fetuses were found to be HBV DNA positive, with all HBV DNA levels under 10(4) copies/ml. HBsAg and HBV DNA detected in the uterus were uncommon and were expressed at low levels. In contract to the case with prenatal statuses, neonatal serologies were more similar to their mothers'. The response rate of vaccination was 95%. Six children for whom immunoprophylaxis failed were born to HBeAg-positive mothers with high HBV DNA levels (>10(8) copies/ml), but only one of them was found to be positive for intrauterine HBV DNA (8.5 × 10(2) copies/ml). The presence of intrauterine hepatitis B antigen and DNA does not indicate postnatal HBV infection and vaccination failure.