To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.