Antibodies against hepatitis E virus (HEV) were detected in sera by a synthetic peptide-based enzyme immunoassay (EIA) from different populations in Venezuela. Antibodies against HEV were found in 1.6% (3/184) of urban pregnant woman (Caracas), in 3.9% (8/204) of rural populations (San Camilo, Edo Apure), and in 5.4% (12/223) of rural Amerindians (Padamo, Edo Amazonas). Positivity was confirmed by a neutralization EIA based on the use of competing soluble free pep-tides. The prevalence of antibodies in the Amerindian group was significantly higher than in urban pregnant women. No relation was found between age and HEV prevalence in rural populations. Three of 21 positive sera were also weakly positive by Western blot for IgM antibodies. This result, together with the low optical density values observed by EIA, suggested that the presence of antibodies in these sera reflects past infections. Based on these results, Venezuela does not seem to be highly endemic for hepatitis E. This is the first report of serological evidence of infection by HEV in South America. © 1994 Wiley-Liss, Inc.