Escherichia coli is a diverse bacterial species that comprises commensal nonpathogenic strains such as E. coli K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic E. coli strain chi7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-E. coli K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and E. coli iro locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain chi7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in E. coli K-12. These E. coli conserved genes are likely to be highly expressed in vivo and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression and physiology of a pathogenic E. coli strain in a natural animal host during the infectious process.