Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerue 01 biotype El Tor strains by ammonium sulfate precipitation. The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity. The toxin formed a high molecular-mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated. The addition of the El Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels, which had long lifetimes at small voltages. The hemolysin channel had a single-channel conductance of 350 pS in 1 M KC1. These results defined hemolysin (HlyA) from V choleme as a channel-forming component with properties similar to other cytolytic toxins. The long lifetime of the channel suggested that the channel-forming oligomer did not show a rapid association/dissociation reaction. At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition. The single-channel conductance of the hemolysin (HlyA) from V cholerue 0 1 biotype El Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm. The hemolysin channel of V cholerue was found to be moderately anion-selective. The pore-forming properties of hemolysin (HlyA) from V cholerue 0 1 biotype El Tor were compared with those of aerolysin of Aeromonus sobriu and atoxin from Staphylococcus uureus. All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion-selective channels.Keywords: hemolysin; ion channel; toxin oligomer; lipid bilayer membrane; Vibrio choleru biotype El Tor.The Gram-negative Vibrio cholerue is an intestinal pathogen that is non-invasive and adheres to the apical side of the small intestine [I, 21. The epidemic qualities of this pathogen have recently been dramatically underscore by the reappearance of cholera in the Western Hemisphere after more than 100 years and the emergence of a new strain, the V cholerae 0 -1 39 Bengal strain, that appeared in India and spread rapidly to countries surrounding the Indian Ocean [3]. Virulence factors elaborated by Y cholerae include cholera toxin, colonization factor, toxinco-regulated pilus, zona occuledens toxin and the regulatory elements ToxR, ToxS and ToxT [2].To establish an infection, V cholerue colonizes the intestinal surface and releases cholera toxin composed of two subunits [4, 51. The binding component ( 5 B subunits) binds to the surface and facilitates the entry of the A subunit into the epithelial cells. The A subunit effects an increase of the intracellular level of cyclic AMP. The high level of intracellular AMP stimulates the secretion of chloride and bicarbonate out of the cell and leads to diarrhoea because of the severe loss of isotonic body fluid [I, 21. Chol...