Hemolysins purified from non-O1 Vibrio cholerae (non-O1 hemolysin) and a Vibrio cholerae O1, biotype El Tor (El Tor hemolysin) were investigated for their homology. The hemolysins were isolated from the culture supernatant fluids by ammonium sulfate precipitation and gel filtration on Sephadex G-100 columns. The purified hemolysins gave single bands with an identical mobility on conventional polyacrylamide gel disc electrophoresis. The molecular weights of the non-O1 and El Tor hemolysins were estimated to be about 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid compositions of the hemolysins were very similar. The specific activities of the hemolysins were identical, and both hemolysins were neutralized to the same extent with antisera against the homologous and heterologous hemolysins. Ouchterlony double immunodiffusion tests with both hemolysins and antihemolysin serum gave a common (fused) precipitin line. These data indicate that the non-O1 hemolysin is biologically, physicochemically, and immunologically indistinguishable from the El Tor hemolysin.