A study was carried out on the effect of a number of inhibitors as well as of anaerobiosis on the production of superoxide (O2 minus) by human granulocytes. Several metabolic inhibitors, including bazide, cyanide, antimycin A, and 2,4-dinitrophenol had no effect on O2 minus production. Methimazole, which inhibits myeloperoxidase, was also without effect. The sulfhydryl reagents N-ethylmalemide and iodoacetamide greatly inhibited O2 minus production. O2 minus production did not take place under N2. Agents that affect cell motility were also examined. Colchicine and vinblastine, alkaloids which affect microtubule function, had little effect on granulycote O2 minus production. On the other hand, cytochalasin B, a fungal antimetabolite thought to affect microfilaments, inhibited O2 minus production by resting granulocytes and stimulated its production by cells incubated with bacteria. The mechanism and significance of this effect remain to be elucidated.