N-ethylmaleimide, divalent cations, ethylene glycol bis (β aminoethyl ether) N,N,N′,N′,-tetraacetate, 2-deoxyglucose, cyanide, and dinitrophenol were examined for their effect on the ability of guinea pig granulocytes to generate superoxide (O₂⁻) when stimulated by digitonin. N-ethylmaleimide (1 mM) inhibits only when added before complete activation of the O₂⁻ generating system, and at lower concentrations (0.05-0.2 mM) slows the activation process. Ca⁺⁺ is required for maximum O₂⁻ generation, and Mg⁺⁺ decreases the amount of Ca⁺⁺ required. Ethylene glycol bis (β aminoethyl ether) N,N,N′,N′,-tetraacetate (10 mM) inhibits only if added before complete activation. Incubation of cells in 2-DOG causes a time- and concentration-dependent inhibition of O₂⁻ generation. It also increases the time required for activation of this system. Cyanide and dinitrophenol increase the rate of O₂⁻ production. However, when these compounds are added to cells whose O₂⁻ production is partially inhibited by incubation in 2-deoxyglucose, complete inhibition results. If cyanide or dinitrophenol is added after activation of 2-deoxyglucose-treated cells, no further inhibition occurs. On the basis of the above results, we conclude that the activation of the O₂⁻ generating system is N-ethylmaleimide sensitive, Ca⁺⁺ dependent, and energy requiring, but that the activity of the enzyme system in the cell is not.