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Antibody to hepatitis E virus (anti-HEV) is prevalent in Western countries, where clinical hepatitis E is rarely reported. The aim of this study was to determine the prevalence of anti-HEV among Danish blood donors and Danish farmers. In addition, we compared the prevalence among 2 sets of serum samples obtained from blood donors 20 years apart.Samples from 291 Danish farmers and 169 blood donors that were collected in 1983 and samples from 461 blood donors that were collected in 2003 were tested for anti-HEV. Relevant information on HEV exposure was collected by self-administered questionnaire.Anti-HEV testing was performed on samples after 20 years of storage at −20°C. The prevalence of anti-HEV was 50.4% among farmers and 32.9% among donors in 1983 and 20.6% among donors in 2003 (P<.05). Presence of anti-HEV was significantly correlated with increasing age in all 3 groups (P<.05). Among donors who had serum samples obtained in 2003, age, contact with horses, and the presence of antibody to hepatitis A virus were associated with the presence of anti-HEV in multivariate analysis. Among farmers, only age was independently associated with the presence of anti-HEV.Anti-HEV was highly prevalent among Danes but has decreased in prevalence over the past 50 years. Our study supports the hypothesis that HEV infection in Denmark may be an asymptomatic zoonotic infection.