We have recently reported the secretion of a 107K polypeptide by an E. coli strain containing the haemolytic plasmid pHly167 (Mackman and Holland 1984). In this paper we show that a large number of haemolytic E. coli strains, apparently including both plasmid and chromosomally located haemolysin genes, secrete similar large molecular weight proteins. Partial purification of one haemolysin suggests that activity co-purifies with a 107K polypeptide. These results were confirmed by cloning the corresponding haemolysin determinant in the form of a recombinant plasmid pLG570, containing chromosomal DNA prepared from a human isolate of E. coli, LE2001. Tn5 was used as a mutagen to localize the haemolysin genes to a 7-kilobase region of pLG570. Structural and export functions were identified by assaying cell sonicates of non-haemolytic mutants. At least one structural gene was identified which coded for a 107K polypeptide. Insertions into this gene completely eliminated haemolysin activity and resulted in truncation of the 107K protein whereas insertions into the adjacent 4-kb region resulted in intracellular haemolytic activity. This internal haemolysin appeared to accumulate in the periplasm which suggests that factors encoded by the 4-kb region are involved in exporting the 107K polypeptide across the outer membrane.