Cytosolic low molecular components in guinea pig neutrophils were examined for the activity to enhance superoxide anion (O2-)-generating NADPH oxidase activity. A component was separated by Sephadex G-25 gel filtration from high molecular weight components, the latter revealed NADPH oxidase activity in a cell-free system in combination with the membrane fraction and arachidonic acid. Addition of this cytosolic low molecular weight component to the cell-free system significantly enhanced NADPH oxidase activity, though this component did not substitute the high molecular weight components in constituting the system. The low molecular weight NADPH oxidase activation factor (LMWAF) found here was not of protein nature, since protease treatment failed to reduce its activity. This factor did not contain phosphate, and was neither flavin nor guanine nucleotide. Though LMWAF was extractable with chloroform-methanol, it was not identical with diacylglycerol.