During an investigation of a hepatitis outbreak occurring in a small village in the Brazilian Amazon, serum samples from 16 recent hepatitis cases and 66 of their asymptomatic relatives were tested for the presence of hepatitis A, B, C, and E markers. Sanitation is poor and organized disposal of sewage is absent in the village. Two of the 16 hepatitis cases were non-A, non-B and non-C hepatitis, but their sera reacted to hepatitis E antibodies of the IgG class (anti-HEV). Likewise, sera from seven of the 66 asymptomatic relatives were positive for anti-HEV. Four of the nine anti-HEV positive sera had their reactivity confirmed by a neutralization test using synthetic peptides based on the nucleotide sequences of open reading frames 2 (ORF2) and 3 (ORF3) encoded in the HEV genome. To our knowledge, this is the first report suggesting the occurrence of acute E hepatitis cases in the Brazilian Amazon.