Haemolytic toxins belong to one of several classes of virulence factors that contribute to bacterial pathogenicity. The non-pathogenic Escherichia coli K-12 laboratory strain was considered for years to be non-haemolytic. However, overproduction of several transcriptional regulators induced the appearance of a haemolytic activity that is absent under usual laboratory growth conditions. In this work, we have cloned and characterized an E. coli K-12 gene, sheA, whose overexpression results in a haemolytic phenotype. It maps to min 27 on the genetic map, and codes for a 34-kDa polypeptide with at least one putative transmembrane segment. This polypeptide, which has neither signal peptide nor other known secretory motifs, is secreted to the medium during the exponential growth phase. In vitro coupled transcription/translation assays, using a plasmid carrying only the sheA gene as template, resulted in the production of a polypeptide with haemolytic activity per se. Our results demonstrate that the sheA gene actually encodes the E. coli K-12 chromosomal haemolysin. The SheA haemolysin does not resemble other known cytolytic toxins, and it may represent the prototype of a novel family, as suggested by the presence of homologues in several E. coli pathogenic strains and in Shigella flexneri.