The extracellular hemolysin from Gardnerella vaginalis (GVH) was purified 88-fold and partially characterized. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 59 kD. The hemolysin is most active at pH 6. The mechanism of hemolysis involves one temperature-dependent binding step followed by cell lysis. GVH does not act like an enzyme but is firmly bound to the erythrocyte membrane and used up during the reaction. Osmotic protectants like sucrose and dextran 4 (4000-6000 D) do not protect the toxin-treated erythrocytes from lysis, but dextran 8 (8000-12,000 D) provides full protection. GVH is inactivated by cholesterol, alpha- and beta-glucosidase. A partial inactivation was found with alpha-mannosidase and N-acetylglucosaminidase. Human lymphocytes are about 100 times less susceptible than human erythrocytes. The specificity of GVH to human erythrocytes is very high. Red cells from monkey, horse, cow, sheep, and rabbit show less than 1% of human erythrocyte susceptibility.