Previous studies reveal that supplementation of human diets with gamma-linolenic acid (GLA) reduces the generation of lipid mediators of inflammation and attenuates clinical symptoms of chronic inflammatory disorders such as rheumatoid arthritis. However, we have shown that supplementation with this same fatty acid also causes a marked increase in serum arachidonate (AA) levels, a potentially harmful side effect. The objective of this study was to design a supplementation strategy that maintained the capacity of GLA to reduce lipid mediators without causing elevations in serum AA levels. Initial in vitro studies utilizing HEP-G2 liver cells revealed that addition of eicosapentaenoic acid (EPA) blocked Delta-5-desaturase activity, the terminal enzymatic step in AA synthesis. To test the in vivo effects of a GLA and EPA combination in humans, adult volunteers consuming controlled diets supplemented these diets with 3.0 g/d of GLA and EPA. This supplementation strategy significantly increased serum levels of EPA, but did not increase AA levels. EPA and the elongation product of GLA, dihomo-gamma-linolenic acid (DGLA) levels in neutrophil glycerolipids increased significantly during the 3-wk supplementation period. Neutrophils isolated from volunteers fed diets supplemented with GLA and EPA released similar quantities of AA, but synthesized significantly lower quantities of leukotrienes compared with their neutrophils before supplementation. This study revealed that a GLA and EPA supplement combination may be utilized to reduce the synthesis of proinflammatory AA metabolites, and importantly, not induce potentially harmful increases in serum AA levels.