The effect of Zn2+ on hemolysis induced by the thermostable direct hemolysin from Vibrio parahaemolyticus (vibriolysin) streptolysin O, and Triton X-100 was studied. We found that in certain buffers, such as tris(hydroxymethyl)-aminomethane-hydrochloride, boric acid-borax, and N-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-sodium hydroxide, hemoglobins released from erythrocytes were easily precipitated by addition of Zn2+, thus resulting in a false inhibition of hemolysin by Zn2+ when hemolysis was assayed by measuring absorbance at 540 nm of released hemoglobins. Under experimental conditions in which hemoglobin was not precipitated, hemolysis induced by streptolysin O was inhibited by Zn2+, whereas that induced by vibriolysin and Triton X-100 was not. Thus, we concluded that the mode of inhibitory action of Zn2+ on hemolysis was not due to a reversible alteration in the state of the lipid bilayer of erythrocytes membranes as proposed by Avigad and Bernheimer (Infect. Immun. 13:1378-1381, 1976).