Production of the siderophore yersiniabactin is associated with virulence in Yersinia species. The genes for biosynthesis and uptake of yersiniabactin are located on a high-pathogenicity island (HPI). The distribution and functioning of the Yersinia HPI were assessed in different Enterobacteriaceae strains isolated from blood cultures and urine samples. In total, 550 clinical isolates from 10 Enterobacteriaceae species were investigated by polymerase chain reaction and DNA hybridization. The Yersinia HPI was most prevalent in Escherichia coli (overall prevalence, 72.3%) and, to a lesser extent, in Klebsiella oxytoca (58.3%), Citrobacter species (25%), Klebsiella pneumonia (17.7%), and Enterobacter species (12.2%). The production of the siderophore yersiniabactin was also demonstrated in these HPI-positive strains by use of a reporter gene bioassay. These findings indicate that the HPI of Yersinia is distributed and functions in clinical specimens of different Enterobacteriaceae species.