Avian pathogenic Escherichia coli (APEC) is an economically important respiratory pathogen of chickens worldwide. Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection. A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure. Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes iss (increased serum survival) and tsh (temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid. These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions. Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and iss were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence. In contrast, the presence of tsh was not necessary for high levels of virulence. Thus, both the aerobactin operon and iss are associated with virulence in APEC after exposure by the natural route of infection. The similarities between APEC and extraintestinal E. coli infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids.