Combined passive and active immunization for newborns very effectively prevents perinatal hepatitis B virus (HBV) infections. In the Netherlands, babies born to hepatitis B surface antigen (HBsAg)-positive women receive passive immunization with hepatitis B and at least three active HBsAg vaccinations. Serological testing for the presence of HBV markers was offered for all infants born to HBsAg-positive mothers between January 2003 and July 2007, after completion of their vaccination schedule. About 75% of the infants (n = 1743) completed their HB-vaccination schedule and participated in the serologic evaluation. Twelve of them (0.7%) were found to be HBV infected. Furthermore, we identified three older children with high levels of anti-HBc, anti-HBs and anti-HBe, while they were HBsAg and HBV DNA negative. This serologic profile is evidence for a resolved HBV infection. In the group of older children (1.5-5 years of age, n = 728), about half of the HBV-infected children (3 of 7) had already cleared their infection at the time of sampling. For a proper evaluation of the efficacy of a new intervention programme to prevent vertical HBV transmission, it is also important to analyse the HBV markers in serum collected when the children are older than 1.5 years. In a programmatic setting, all children born to HBV-infected mothers should be tested not only for the level of anti-HBs but also for the absence of HBsAg, because 2 of the 12 HBV-infected children (17%) had a high level of anti-HBs.