Site-directed mutagenesis of the TGC codon in a cloned streptolysin O (SLO) gene exchanged the single Cys residue in SLO for either Ala or Ser. The parent wild-type SLO (SLO.Cys-530) and the SLO.Ala-530 and SLO.Ser-530 mutant toxins, expressed in Escherichia coli, were purified and analyzed. Wild-type SLO.Cys-530 and the SLO.Ala-530 mutant showed no significant differences in their specific hemolytic activities, while the SLO.Ser-530 mutant had a reduced (ca. 25%), but still considerable, specific hemolytic activity as compared with that of wild-type SLO. The parent and mutant toxins extracted from lysed erythrocyte membranes had similar sedimentation profiles on sucrose density gradients, suggesting that the mutations did not affect the ability of SLO to form oligomers in membranes. These results show that the widely held assumption that the in vitro cytolytic activity of SLO requires an essential Cys residue is not true.