When the steady-state concentrations of peroxide in prostaglandin H synthase assay systems was lowered by added glutathione peroxidase, several agents (meclofenamic acid, mefenamic acid, acetamidophenol and phenylbutazone) became more potent inhibitors of the prostaglandin-forming cyclooxygenase reaction. Paradoxically, these agents stimulated oxygen incorporation in the absence of added peroxidase. On the other hand, dithiothreitol, ibuprofen, flurbiprofen and indomethacin all inhibited the reaction in a dose-dependent manner, and their inhibitory potencies were unaffected by the action of glutathione peroxidase. Aspirin, dl-gamma-tocopherol and salicylic acid were not inhibitory (without preincubation) under the assay conditions employed in this study. The findings demonstrate that the potencies of some anti-inflammatory agents may be diminished by high local peroxide concentrations.