DNA sequences similar to those of the Escherichia coli hemolysin genes were detected among uropathogenic isolates of Proteus vulgaris and Morganella morganii by using the Southern blotting technique and hly gene-specific DNA probe. Immunoblotting revealed that among the hemolytic P. vulgaris and M. morganii isolates there was expressed a polypeptide species similar in molecular size (110 kilodaltons) and antigenicity to Escherichia coli HlyA. A plasmid-mediated P. vulgaris hemolysin determinant identified by Southern blotting analysis was molecularly cloned, and the recombinant plasmid (pWPV100) was characterized by restriction endonuclease fragment mapping. A second recombinant library of genomic DNA prepared from a hemolytic, urinary tract isolate of Proteus mirabilis was constructed in E. coli. A 5.5-kilobase XhoI fragment encoding an extracellular hemolytic activity was molecularly cloned (pWPM100), and this plasmid was subjected to transposon-mediated mutagenesis with TnphoA. The P. mirabilis hemolytic phenotype was determined to be encoded by a polypeptide species (HpmA) with an estimated molecular size of 140 kilodaltons based on minicell polypeptide analysis of pWPM100 and its mutant derivatives. Southern blotting analysis with a HpmA-specific DNA probe revealed that this novel determinant is commonly found in both Proteus species but is not present in hemolytic isolates of M. morganii, E. coli, Citrobacter freundii, and Serratia marcescens.