Plasmid hemolysin (hly) determinants have been shown previously to comprise three cistrons (hlyA, hlyB, hlyC), coding for the synthesis and transport of hemolysin. Using recombinant plasmids as specific probes for these cistrons, we were able to analyze the chromosomal hly determinants of nine Escherichia coli strains which belonged to serotypes O4, O6, O18, and O75 and were isolated from urinary tract infections and fecal flora. The chromosomal hly genes shared extensive sequence homology with the cloned plasmid hly determinant. Nevertheless, small differences were observed, and these were found to lie mainly within cistron A (hlyA), which has been shown to determine the hemolysin protein itself. These fine variations were not specific for the O-serotype.