The role of rats in human hepatitis E virus (HEV) infections remains controversial. A genetically distinct HEV was recently isolated from rats in Germany, and its genome was sequenced. We have isolated a genetically similar HEV from urban rats in Los Angeles, California, USA, and characterized its ability to infect laboratory rats and nonhuman primates. Two strains of HEV were isolated from serum samples of 134 wild rats that had a seroprevalence of antibodies against HEV of ≈80%. Virus was transmissible to seronegative Sprague-Dawley rats, but transmission was spotty and magnitude and duration of infection were not robust. Viremia was higher in nude rats. Serologic analysis and reverse transcription PCR were comparably sensitive in detecting infection. The sequence of the Los Angeles virus was virtually identical to that of isolates from Germany. Rat HEV was not transmissible to rhesus monkeys, suggesting that it is not a source of human infection.