The inhibitory effects of sulfasalazine, some sulfasalazine-related compounds and indomethacin on superoxide production by human polymorphonuclear (PMN) leukocytes were studied. The inhibition of the chemotactic peptide (FMLP)-induced superoxide production, which is membrane receptor-mediated, was strongly dependent on the concentration both of the secretory stimulus and of the test compounds, indicating an interaction between the receptor and the test compound. Furthermore, a positive correlation was found between the lipophilicity of the compound and the degree of inhibition. However, when the receptor was by-passed by direct activation of the receptor-linked G protein by the use of fluoride ions as secretory stimuli, the test compounds still inhibited superoxide production. On the other hand, superoxide production by cells stimulated with phorbol ester was not inhibited by the test compounds. Furthermore, the production of phosphatidic acid was decreased in the presence of sulfasalazine, indicating impaired phosphoinositide metabolism. The inhibition of this metabolism was not due to increased intracellular concentrations of cyclic AMP, although sulfasalazine did inhibit cyclic nucleotide phosphodiesterase. We conclude that sulfasalazine attenuates superoxide production by PMN leukocytes at a post-receptor site of action at a step before the activation of protein kinase C, possibly by interfering with the phosphoinositide metabolism but independent of cyclic AMP.