Australia is a unique setting for a Japanese encephalitis virus (JEV) outbreak since it is home to a large array of fauna not seen elsewhere in the world. While waterbirds and pigs are known reservoirs for the virus, limited information is available on the capacity for other endemic animal species to act as maintenance/amplifying hosts. In 1999 and 2000 experimental infection studies were performed at the CSIRO Australian Animal Health Laboratory to determine if Eastern grey kangaroos, Agile wallabies, Tamar Wallabies, European Rabbits and Brushtail possums support JEV replication. Groups of 4–5 animals were subcutaneously inoculated with 105 TCID50 units of JEV isolate 4054. Serum was collected daily for 7 days post-infection (dpi), with subsequent sampling timepoints at 10, 14 and 18–23dpi. Virus isolation was performed on 1–7 dpi serum in cell culture. Serum collected from rabbits at 3dpi was also assessed by suckling mouse inoculation (SMIC). Antibody response was measured by JE competitive cELISA and plaque reduction neutralisation test (PRNT). Brief low-level viremia (up to 101.6 TCID50/ml) was detected in 2/5 Tammar and Agile wallabies, but not in Eastern grey kangaroos. Seroconversion was detected in 1–2 animals of each macropod species, with weak and transient neutralising antibody responses measured in PRNT. All five rabbits seroconverted to JEV by 10 dpi, with low level viraemia identified in one rabbit by SMIC only. In contrast viraemia was detected in 4/4 brushtail possums over 2-3 days (peak titre 103 TCID50/ml), with all possums developing a strong neutralising antibody response from 14dpi onwards. Subsequent sequencing of JEV isolate 4054 shows it belongs to Genotype II, and clusters with sentinel pig isolates from the 1998 Australian outbreak. This work furthers our understanding of JEV host ecology and identifies brushtail possums as potential maintenance hosts for JEV in peri-urban habitats.