Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10.5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.