Bovine erythrocytes (RBCs) exposed to Moraxella bovis culture supernatants exhibited rapid leakage of intracellular K+ (95% in 10 min), slower cell swelling (1.20-fold increase in mean corpuscular volume in 20 min), and subsequent lysis (76% leakage of hemoglobin in 25 min). Incubation media made hypertonic by the addition of 75 mM carbohydrates with molecular diameters of 0.72 to 1.32 nm prevented hemolysin-induced RBC swelling, but incubation media made hypertonic by the addition of carbohydrates with molecular diameters of less than 0.72 nm did not protect against hemolysin-induced RBC swelling. Raffinose (75 mM; molecular diameter, 1.14 nm) did not block hemolysin-induced K+ leakage but did block hemolysis. These findings support the hypothesis that hemolysin-induced lysis occurs by colloid-osmotic swelling and are compatible with M. bovis hemolysin acting as a pore-forming cytolysin. Assuming that M. bovis hemolysin acts as a transmembrane molecular sieve, then the functional size of the hemolysin transmembrane pores in bovine RBCs is approximately 0.9 nm, the molecular size of sucrose. Hemolytic activity was inhibited by the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), but hemolysin-induced K+ leakage was not affected by EGTA.