Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an O2- -generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of O2 to O2- by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the O2- -generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither O2- nor H2O2. The amount of such a non-O2- -producing O2 consumption often reached twice as much as that of O2- production. For the activation of the O2- -generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-O2- -producing O2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the O2- -generating system, especially that by saturated fatty acids.