Decades after smallpox was eradicated and vaccination discontinued, the level of residual immunity in today's population is largely unknown. This study describes an epidemiological assessment in Italians of antibodies against the intracellular mature virus (IMV) and extracellular envelope virus (EEV) forms of Vaccinia virus. Serum samples (n=642) were taken in 1993 and 2003 from people between 11 and 102 years old. Most citizens >27 years old were positive for antibodies to IMV and EEV. These antibodies were long-lasting and similar titres were present in citizens between 30 and 100 years old. Serum samples from 1993 and 2003 displayed very similar EEV-and IMV-specific antibody titres. By using these data and demographic considerations, it was predicted that, in 2003, 46 % of the Italian population were positive for both IMV and EEV, 42 % were negative for both and 12 % were positive for one antigen.Variola virus, the causative agent of smallpox, was eradicated in 1977 after widespread vaccination with Vaccinia virus (VACV) (Fenner et al., 1988). In Italy and many other European countries, smallpox vaccination used predominantly VACV strain Lister/Elstree. Vaccination was compulsory in Italy until 1976(Tagarelli et al., 2004 although, after 1974, public-health services were less stringent in their vaccination policy. Mass smallpox vaccination was abandoned in most European countries in the early 1970s and production of the vaccine was discontinued in the early 1980s.Understanding of the immune responses induced after vaccination with VACV is incomplete and concern about the deliberate release of Variola virus (Smith & McFadden, 2002) has prompted investigation of how much immunity remains in populations today. It was generally accepted that vaccination would protect most vaccinees for 5-10 years (Fenner et al., 1988), although the World Health Organization (WHO) recommended revaccination every 3 years for those in areas where smallpox was endemic. Recent studies have shown that humoral (Crotty et al., 2003;Frey et al., 2003;Gallwitz et al., 2003;Hammarlund et al., 2003;Hatakeyama et al., 2005;Viner & Isaacs, 2005) and cellular (Hammarlund et al., 2003;Amara et al., 2004;Combadiere et al., 2004;Kennedy et al., 2004) forms of immunity are long-lived after VACV immunization, but it is unknown whether this remaining immunity will protect against smallpox. Although analysis of historical data suggests that protection after vaccination might last for several decades (Hanna & Baxby, 2002;Eichner, 2003a), the last smallpox fatality, in Birmingham, UK, in 1978, occurred despite the patient having been vaccinated twice previously, once as a child and once 12 years before contracting the disease (Shooter, 1980). VACV produces two morphologically and antigenically distinct infectious forms of virus, called intracellular mature virus (IMV) and extracellular envelope virus (EEV) . Most infectious virus particles remain in the cell as IMV until cell lysis; this form is very stable and so is likely to be responsible for host-to...