Moraxella bovis (M. bovis) is the etiologic agent of infectious bovine keratoconjunctivitis and M. bovis hemolysin is believed to be an important virulence factor. Two strains of M. bovis were compared, Epp 63(300) (Epp), a known virulent and hemolytic strain, and IBH 63 (IBH), a known avirulent and nonhemolytic strain. Sterile 10-fold (10x) supernatant concentrates were obtained from cultures grown in TSB broth with 10 mM CaCl2. Supernatant hemolysin titers for Epp, were 1:1024 and 1:8192 for unconcentrated (1x) and 10x, respectively. Supernatant cytotoxin titers to bovine mononuclear cells were 1:32 and 1:128 for 1x and 10x, respectively, for Epp. Cytolytic (hemolytic and cytotoxic) activities declined 10-fold but were still measurable for > 1 wk at 4 degrees C. Both activities were inactivated by trypsin and by heating at 56 degrees C for 20 min. A cytotoxic effect was observed on cultured bovine and ovine corneal epithelial cells with Epp. All cytolytic effects were neutralized with antiserum to 10x Epp. No cytolytic activities were detected for 10x IBH. SDS-PAGE electrophoresis and related immunoblots indicate a high molecular weight protein at 110 kDa for the 10x Epp preparation when stained with silver or probed with monoclonal antibodies to the E. coli alpha hemolysin. No 110 kDa band is observed for 10x IBH. These data suggest that hemolytic and cytotoxic activities are important in the pathogenesis of infectious bovine keratoconjunctivitis and identify the protein as a possible RTX related toxin of 110 kDa. Stability of the M. bovis cytolysin for > 1 week should allow further characterization and purification of the protein.