Based on computer analysis of hydrophobicity and prediction of secondary structures for the full-length putative proteins encoded by open reading frame-1 (ORF-1), ORF-2 and ORF-3 of hepatitis E virus (HEV), we selected antigenic regions with hydrophilicity, beta-turn, and beta-sheet, and synthesized 7 peptides of possible epitope-containing regions of the polypeptide encoded by all 3 ORFs of HEV genomic RNA by Merrifield's method of solid-phase synthesis. The synthetic peptides were screened and identified by solid-phase enzyme-linked immunosorbent assay (ELISA). Three of the peptides (EH174 from ORF-1, EH286 from ORF-2 and EH362 from ORF-3) showed antigenic activity and possible application for the development of anti-HEV test kits (the peptide-based ELISA). The laboratory experiments and clinical trials showed that the kits, using a set of 3 synthetic HEV peptides as coating antigens, were of high specificity and exhibited good reproducibility. The small-scale seroepidemiological survey indicated high seroprevalence (14.3%) of anti-HEV in Tibetan populations. Additionally, the results also demonstrated good agreement with clinical findings, suggesting that the test kits will be of major use for immunodiagnosis and seroepidemiological surveys of HEV infection.