Avian hepatitis E virus (HEV), a novel virus identified from chickens with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human HEV. In order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated, as HEV cannot be propagated in vitro. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock as a 10% suspension of positive fecal and bile samples in phosphate-buffered saline. The infectivity titer of this infectious stock was determined by inoculating 1-week-old SPF chickens intravenously with 200 microl of each of serial 10-fold dilutions (10(-2) to 10(-6)) of the avian HEV stock (two chickens were inoculated with each dilution). All chickens inoculated with the 10(-2) to 10(-4) dilutions of the infectious stock and one of the two chickens inoculated with the 10(-5) dilution, but neither of the chickens inoculated with the 10(-6) dilution, became seropositive for anti-avian HEV antibody at 4 weeks postinoculation (wpi). Two serologically negative contact control chickens housed together with chickens inoculated with the 10(-2) dilution also seroconverted at 8 wpi. Viremia and shedding of virus in feces were variable in chickens inoculated with the 10(-2) to 10(-5) dilutions but were not detectable in those inoculated with the 10(-6) dilution. The infectivity titer of the infectious avian HEV stock was determined to be 5 x 10(5) 50% chicken infectious doses (CID(50)) per ml. Eight 1-week-old turkeys were intravenously inoculated with 10(5) CID(50) of avian HEV, and another group of nine turkeys were not inoculated and were used as controls. The inoculated turkeys seroconverted at 4 to 8 wpi. In the inoculated turkeys, viremia was detected at 2 to 6 wpi and shedding of virus in feces was detected at 4 to 7 wpi. A serologically negative contact control turkey housed together with the inoculated ones also became infected through direct contact. This is the first demonstration of cross-species infection by avian HEV.