The aim of this study was to determine if rhesus monkeys infected with one isolate of hepatitis E virus (HEV) were immune to subsequent challenge with other isolates of the virus. Three epidemic and one sporadic Indian HEV isolates were employed in the study. The interval between primary inoculation and challenge varied from 1 year and 6 months to 2 years and 9 months. Evidence of HEV infection was ascertained by rise in serum alanine transaminase (ALT) levels and/or seroconversion to antibodies to HEV (anti-HEV), and the presence of HEV-RNA in the bile or faeces of the infected monkeys. No evidence for multiplication of virus in monkeys challenged with different HEV isolates was obtained. These results show that immunity generated by one isolate of HEV protects against different isolates of hepatitis E virus. © 1995 Wiley-Liss, Inc.