The hemolytic activity of α-toxin ofMicrococcus pyogenesvar.aureushas been shown to be maximal at pH 6.8–7.0 in phosphate-buffered saline. Within the limits of 25 to 75% hemolysis there is a linear relationship between lysin concentration and extent of hemolysis after incubation for 15 minutes. Citrate and glucose have been shown to inhibit hemolysis by α-toxin. The manner in which the factors studied affect determination of α-toxin activity by a direct hemolysis procedure is discussed.