Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0x10(7) copies ml-1, genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4x10(4) copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1x10(5) copies ml-1 on day 60. When 8.6x10(5) copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6x10(7) copies ml-1 on day 60. HEV incubated at temperatures higher than 70 degrees C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 degrees C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.