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A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202‐312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.