A prospective seroepidemiological survey was carried out in Luxembourg in 2000–2001 to determine the antibody status of the Luxembourg population against hepatitis A virus (HAV) and hepatitis B virus (HBV). One of the objectives of this survey was to assess the impact of the hepatitis B vaccination programme, which started in May 1996 and included a catch-up campaign for all adolescents aged 12–15 years. Venous blood from 2679 individuals was screened for the presence of antibodies to HAV antigen and antibodies to hepatitis B surface antigen (anti-HBs) using an enzyme immunoassay. Samples positive for anti-HBs were tested for antibody to hepatitis B core antigen (anti-HBc) using a chemiluminiscent microparticle immunoassay to distinguish between individuals with past exposure to vaccine or natural infection. The estimated age-standardized anti-HAV seroprevalence was 42.0% [95% confidence interval (CI) 39.8–44.1] in the population >4 years of age. Seroprevalence was age-dependent and highest in adult immigrants from Portugal and the former Yugoslavia. The age-standardized prevalence of anti-HBs and anti-HBc was estimated at 19.7% (95% CI 18.1–21.3) and 3.16% (95% CI 2.2–4.1) respectively. Anti-HBs seroprevalence exceeding 50% was found in the cohorts targeted by the routine hepatitis B vaccination programme, which started in 1996. Our study illustrates that most young people in Luxembourg are susceptible to HAV infection and that the hepatitis B vaccination programme is having a substantial impact on population immunity in children and teenagers.