Entero-invasive Escherichia coli (EIEC) and shigellae were tested for contact-haemolysin (CH) with red blood cells (RBCs) of guinea-pig, rabbit, rat, mouse, monkey, man, sheep and chicken; all bacteria showed the best lysis with guinea-pig RBCs. The best culture medium for CH activity of shigellae was tryptic soy broth, and for EIEC it was casamino acid-yeast extract broth with 1 mM CaCl2. CH production by all species was best at the slightly alkaline pH which is optimal for growth; it was also dependent on the presence of a large (140-Mda) plasmid. Pre-treatment of bacteria with homologous antisera inhibited CH activity. Various treatments of bacterial cells and RBCs suggested that CH may be a protein molecule, and that a chitotriose-like moiety may serve as CH receptor. RBCs that were incubated with bacteria at 4 degrees C, or with heat-killed bacteria at 37 degrees C, were not lysed; also, isolated cell-surface components (lipopolysaccharide and outer-membrane protein) did not lyse RBCs. This suggests that metabolically active cells are required for CH activity. Production of CH by both EIEC and shigellae is consistent with a common mechanism for the virulence of these organisms.