The Australian National Poliovirus Reference Laboratory at the Victorian Infectious Diseases Reference Laboratory is the World Health Organization designated laboratory for the isolation and testing of poliovirus from clinical specimens within Australia, the Pacific Island countries and Brunei Darussalam. Surveillance for acute flaccid paralysis (AFP) within Australia, the main clinical manifestation of poliomyelitis, is also coordinated at the Victorian Infectious Diseases Reference Laboratory in conjunction with the Australian Paediatric Surveillance Unit. The annual non-polio acute flaccid paralysis rate after classification of cases by the Polio Expert Committee was 1.0 per 100,000 population, reaching the expected World Health Organization annual target for a non-polio endemic country. During 2004, 64 specimens from 30 AFP cases were referred to the National Polio Reference Laboratory. A mixture of poliovirus types 1 and 2 was isolated from an infant with AFP from New South Wales. Both isolates tested as Sabin-like and the case was subsequently classified as infant botulism by the Polio Expert Committee. The laboratory isolated adenoviruses from seven AFP cases. A coxsackievirus B5 and an echovirus 18 were identified from a further two AFP cases. During 2004, 1,266 cases of poliomyelitis due to wild poliovirus were reported world-wide. Many of these resulted from wild poliovirus importations, which continued in 2005, including to Indonesia. This highlights the need for maintaining high poliovirus vaccination coverage to prevent the transmission of poliovirus and high quality AFPand laboratory surveillance for the detection of poliomyelitis due to an imported wild poliovirus.