The effects of anti-inflammatory drugs (acetylsalicylic acid, ASA; salicylic acid, SA; indomethacin, IM; hydrocortisone, HC) on the respiratory burst oxidase (NADPH oxidase) from human neutrophils in both whole cell and fully soluble (cell-free) systems were investigated. These drugs were found to inhibit the superoxide generation by human neutrophils exposed to phorbol myristate acetate in a whole cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in cell-free systems. Concentrations of these drugs required for 50% inhibition of the oxidase (ID₅₀) were; ASA (more than 3.0 mM in the whole cell system and 1.35 mM in the cell-free system), SA (more than 3.0 mM in the whole cell system and 1.30 mM in the cell-free system), IM (180 μM in both systems) and HC (50 μM in the whole cell system and 40 μM in the cell-free system). In addition, these drugs time-dependently inhibited the activation of NADPH oxidase in cell-free systems. In the cell-free system, all of the drugs did not change the Km values for NADPH of the oxidase. These results suggest that these anti-inflammatory drugs, especially HC and IM, inhibit the reconstitution (activation) of neurtophil NADPH oxidase enzyme in the cell-free (whole cell) system.