Group B streptococci (GBS) express a β‐haemolysin/cytolysin that contributes to disease pathogenesis. We report an independent discovery and extension of a genetic locus encoding the GBS β‐haemolysin/cytolysin activity. A plasmid library of GBS chromosomal DNA was cloned into Escherichia coli, and a transformant was identified as β‐haemolytic on blood agar. The purified plasmid contained a 4046 bp insert of GBS DNA encoding two complete open reading frames (ORFs). A partial upstream ORF (cylB) and the first complete ORF (cylE) represent the 3′ end of a newly reported genetic locus (cyl) required for GBS haemolysin/cytolysin activity. ORF cylE is predicted to encode a 78.3 kDa protein without GenBank homologies. The GBS DNA fragment also includes a previously unreported ORF, cylF, with homology to bacterial aminomethyltransferases, and the 5′ end of cylH, with homology to 3‐ketoacyl‐ACP synthases. Southern analysis demonstrated that the cyl locus was conserved among GBS of all common serotypes. Targeted plasmid integrational mutagenesis was used to disrupt cylB, cylE, cylF and cylH in three wild‐type GBS strains representing serotypes Ia, III and V. Targeted integrations in cylB, cylF and cylH retaining wild‐type haemolytic activity were identified in all strains. In contrast, targeted integrations in cylE were invariably non‐haemolytic and non‐cytolytic, a finding confirmed by in frame allelic exchange of the cylE gene. The haemolytic/cytolytic activity of the cylE allelic exchange mutants could be restored by reintroduction of cylE on a plasmid vector. Inducible expression of cylE, cylF and cylEF demonstrated that it is CylE that confers haemolytic activity in E. coli. We conclude that cylE probably represents the structural gene for the GBS haemolysin/cytolysin, a novel bacterial toxin.