- The effect of arachidonic acid (AA) on the assembled NADPH oxidase activity in cytoplasmic membranes and in endosomes separated from human neutrophils was studied. These two fractions were separated on a Percoll-sucrose density gradient from PMA-stimulated neutrophils preincubated with fluorescein isothiocyanate-conjugated dextran (FITC-dextran). In both fractions, NADPH oxidase activity could be detected with the addition of NADPH and cytochrome c , indicating the presence of an assembled activated form of the enzyme. Addition of AA at low concentrations ( ED 50 = 1 μ M and 0.1 μM for cytoplasmic membranes and FITC-dextran endosomes, respectively) caused an increase in the activity of the assembled NADPH oxidase found in these fractions. Addition of 10 μM AA to the assembled oxidase in cytoplasmic membranes or endosomes significantly increased the V max (1.37 and 1.45 nmol O 2 /min compared with 2.05 and 2.20 nmol O 2 /min in the absence or presence of AA, respectively) and lowered the K m for NADPH (35 μM and 40 μM compared with 7.5 μM and 7.2 μM in the absence or presence of AA, respectively). These results suggest that AA increases the activity of the assembled NADPH oxidase by elevating the number of its active forms and increasing its affinity to the substrate.