The synthesis and secretion of the toxic exoprotein alpha-haemolysin of E. coli PM152 is coded by the transmissible plasmid pHly152 (41 x 10(6) dalton) as shown by the transformation of the plasmid DNA and the isolation of mutants that are specifically altered in the synthesis and transport of haemolysin. These mutants were obtained by chemical mutagenesis and insertion of the ampicillin transposon (Tn3) into pHly152. Tn3 transposition was also used for the identification and the location of the cistrons on pHly152 essential for haemolysis. The EcoRI and HindIII fragments of the haemolytic plasmid pHly152 were cloned and used for the complementation of the haemolysis negative Tn3 insertion mutants. A DNA segment of 3.2 x 10(6) dalton could be thus identified which consists of at least three clustered cistrons necessary for haemolysis. Two of these cistrons are required for the formation of active haemolysin. At least one other cistron seems to be involved in the secretion of active haemolysin through the outer membrane of E. coli. The gene products determined by these cistrons were identified in minicells of E. coli. Their molecular properties were determined and their possible function in the formation and secretion of haemolysin will be discussed.