- Background: In July 1999, a national toddler-only hepatitis A virus (HAV) vaccination program was introduced in Israel. Passive and active surveillance showed a large reduction in disease rate, but an objective measurement was needed. We hypothesized that toddler's vaccination in a population living in an endemic area would reduce virus circulation, resulting in reduced HAV seropositivity rates in unvaccinated toddlers. Methods: The study was conducted among Bedouin children in southern Israel, for whom HAV vaccine coverage reached 85.5% and 74.9% for first and second HAV vaccine doses, respectively, in 2000. Toddlers received 2 doses of HAV vaccine at 18 and 24 months. Data on vaccine coverage was received from well-baby clinics. Sera were obtained from healthy unvaccinated 16- to 20-month-old toddlers. Anti-HAV immunoglobulin (Ig)G concentrations were tested by enzyme-linked immunosorbent assay. Results: A total of 629 sera were tested (209 obtained in 1991–2000 and 420 obtained in 2001–2002). Seropositivity rates of ≥100 mIU/mL ranged from 16.2% to 19.6% in 1991 through 2000 (children born before immunization program). These rates dropped to 2% in 2001–2002 and to 0% in 2003 through 2007. Furthermore, IgG concentrations were significantly lower (P < 0.001) in samples taken in 2000, only a few months after beginning of vaccination, than in those taken before initiation of the HAV immunization program (1991–1998), suggesting a marked reduction in circulating HAV resulting in natural boosting. Conclusions: Because HAV vaccines are licensed in children ≥12 months old, rates of anti-HAV seropositivity in unvaccinated toddlers can be an objective and sensitive tool to evaluate the effect of immunization program on virus circulation. This method is of special value in communities where no appropriate surveillance is in place.