The prevalence of anti-HEV was assessed in 2,233 subjects aged 20-79 years in the Republic of San Marino in the years 1990-1991. The sera were tested by ELISA and further confirmed by Western blot (WB) analysis. The overall anti-HEV prevalence was 1.5%. A significant trend by age was observed. Anti-HEV prevalence was 0.6% in subjects <30 years and 3.3% in those older than 70 years of age. Family size larger than four persons (OR = 3.8; 95% CI = 1.8-13.2) was the sole independent predictor of anti-HEV positivity in the multivariate analysis. Anti-HAV and anti-HEV prevalences did not show a parallel trend by age. No association was found either between hepatitis E virus (HEV) or hepatitis C virus (HCV) infections. Follow-up samples 5 years apart were available for 38 out of 54 (70%) anti-HEV ELISA-positive subjects. Eight out of 22 (37%) WB-confirmed anti-HEV-positive subjects were still anti-HEV-positive after 5 years. However, anti-HEV remained positive in all but two (75%) of the subjects with WB-confirmed ELISA positivity value of S/CO > or = 2 (cutoff 1.2), but in only 2 out of the 14 subjects (14%) with a WB-confirmed ELISA positivity value of S/CO < 2 (P < 0.005). None of the 16 subjects ELISA-positive but not WB-confirmed was anti-HEV-positive 5 years apart. Therefore, only a relative proportion of subjects once infected with HEV maintain for at least 5 years anti-HEV antibodies.