Arachidonic acid (AA) has been implicated as an important amphiphilic co-factor in the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in neutrophils and reconstituted cell-free systems. To assess the role of AA in the activation of O2- generation in monocytic cells, we studied pre-monocytic U937 cells differentiated with 1,25-(OH)2-vitamin D3 plus interferon-gamma (IFN-gamma). AA dose-dependently enhanced phorbol myristate acetate (PMA)-stimulated O2- generation, with a maximum increase of 4,5-fold, through: (1) a more than 50% reduction of the lag-phase, defined as the time between addition of PMA and detection of O2-; and (2) a more than 60% increase in the constant rate of O2- generation. Reduction of the lag phase was associated with increased protein kinase C (PKC)-independent translocation of the cytosolic subunit of NADPH oxidase p47-phox to the cell membrane, whereas increased generation of O2- correlated with enhanced activation of PKC. The data indicate that AA increases activation of NADPH oxidase by accelerating its assembly and by co-stimulating PKC in monocytic U937 cells.