Serum antibodies to strains of avian paramyxovirus and flavivirus were detected in little blue penguins sampled at Port Campbell and Phillip Island, Victoria, Australia. No antibody to Newcastle disease virus (NDV) was detected in 267 sera collected, although one penguin captured for experimental studies had a hemagglutination-inhibition antibody titer of 24 to this virus. Experimental studies showed that the avian paramyxovirus designated APMV-IM and strain V4 of NDV were non-pathogenic for penguins, although the penguins could have been previously infected with these or similar virus strains. A flavivirus designated Saumarez reef virus, and an unnamed virus isolated from ticks on Macquarie Island, Southern Ocean were pathogenic causing disease and mortality in penguins inoculated with the viruses.