Abstract Although rat hepatitis E virus (HEV) has been identified in wild rats, no cell culture systems for this virus have been established. A recent report suggesting the presence of antibodies against rat HEV in human sera encouraged us to cultivate rat HEV in human cells. When liver homogenates obtained from wild rats (Rattus rattus) in Indonesia were inoculated onto human hepatocarcinoma cells, the rat HEV replicated efficiently in PLC/PRF/5, HuH-7 and HepG2 cells, irrespective of its genetic group (G1–G3). The rat HEV particles released from cultured cells harbored lipid-associated membranes on their surface that were depleted by treatment with detergent and protease, with the buoyant density in sucrose shifting from 1.15–1.16g/ml to 1.27–1.28g/ml. A Northern blotting analysis revealed genomic RNA of 7.0kb and subgenomic RNA of 2.0kb in the infected cells. The subgenomic RNA of G1–G3 each possessed the extreme 5′-end sequence of GUAGC (nt 4933–4937), downstream of the highly conserved sequence of GAAUAACA (nt 4916–4923). The establishment of culture systems for rat HEV would allow for extended studies of the mechanisms of viral replication and functional roles of HEV proteins. Further investigation is required to clarify the zoonotic potential of rat HEV.