Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.