Human neutrophils were treated with streptolysin O in amounts that failed to cause cell lysis or morphologic changes. As a result of this treatment, the neutrophils were unable to respond to chemotactic factors. The inhibition of chemotaxis by streptolysin O was independent of factors in serum and could be prevented by heat inactivation, oxidation, and serum containing antistreptolysin O if used in sufficient titer with prior incubation. In addition to preventing directional mobility of leukocytes, streptolysin O also suppressed the random migration of neutrophils.