Among a large collection of hemolysis-negative mutants obtained by mutagenesis of the Hly plasmid pHly152 with Tn5, we have isolated two classes of mutants which are defective in the transport of hemolysin across the outer membrane. The two cistrons (hylBa and hlyBb) which are affected in these mutants are located adjacent to each other on the hly determinant but are transcribed from different promoters. Recombinant plasmids were constructed which carry the two functions as combined or separated cistrons. These were shown to complement the two types of transport mutants. Studies on the compartmentation of hemolysin in these two classes of mutants indicate that most hemolysin (greater than 70%) in hlyBa mutants is located in the periplasmic space, whereas in hlyBb mutants a larger portion of hemolysin is associated with the outer membrane fraction. The phenotypic appearance of colonies from hlyBb mutants is that of beta-hemolytic Escherichia coli strains, indicating that a substantial portion of hemolysin has already reached the outside of the outer membrane without being released into the medium. Release was achieved readily when hlyBb mutants were complemented with a recombinant plasmid carrying hlyBb.