Cross-neutralization studies on leptospiral hemolysins from strains of two antigenically different serotypes, pomona and canicola, were conducted in sheep. A third strain of serotype hardjo that does not produce hemolysin and is antigenically distinct was included for control purposes. Concentrated hemolysins, prepared from supernatant fluids of canicola or pomona cultures, produced hemolytic anemia in sheep after intravenous injection. Sheep previously infected with hemolysin-producing strains were refractory to effects of homologous or heterologous hemolysins. On the other hand, infection with hardjo did not confer immunity to the action of hemolysins. Hemolysin-neutralizing antibodies were demonstrable in sheep previously infected with pomona or canicola only after challenge with homologous or heterologous hemolysins. Cross-neutralization between two hemolysins were demonstrable in vitro. Hemolysin-neutralizing antibody titers did not correlate with agglutinin titers. Concentrated supernatant fluid of the hardjo culture provoked toxic reactions predominantly in sheep previously infected with pomona or canicola. The causes of these untoward reactions were not determined.