The seroprevalence of antibodies to HEV was determined in three different Italian populations (volunteer blood donors, normal healthy individuals, and intravenous drug users [IVDUs]). The seroprevalence in the populations ranged from 0.74% to 1.94%, with higher rates among IVDUs and individuals over 40 years of age. None of 35 patients diagnosed with acute hepatitis A but 10 of 153 (6.5%) patients with acute nonA, nonB, nonC hepatitis were found to be positive for anti-HEV antibody. One of these antibody positive cases was linked to travel, while the remaining 9 were not associated with travel to endemic areas. These data suggest a possible low circulation of HEV in Italy.