To evaluate the persistence of the immune response in a population of healthy volunteers that had been vaccinated with a new Hansenula polymorpha recombinant hepatitis B vaccine in a previous clinical study 4 years before, we measured the titre of anti-HBs. All, but one of the evaluated volunteers remained protected. None of them had experienced any adverse event related to the vaccine from the moment of immunization, to the present. The vaccine proved to be immunogenic and to confer long-term protection in this group.