There is a significant interest in the interrelationship between long- chain n-3 and n-6 fatty acids due to their ability to modulate eicosanoid production. In general, the intake of arachidonic acid (AA) results in enhanced eicosanoid production, whereas n-3 polyunsaturated fatty acids (PUFA) decrease the production of eicosanoids from AA. The purpose of this study was to investigate whether the effects of dietary AA on eicosanoid production in the rat were correlated with the AA and EPA levels in platelets and aorta (eicosanoid-producing tissues). Four groups of male Sprague-Dawley rats were fed a high-fat diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (approximately 100 mg/day of EPA + DHA) for 24 d. During the last 10 d, the four groups were orally supplemented with 0, 30, 60, and 90 mg/day of ethyl arachidonate. A further group of rats was fed a control diet (without long-chain n-3 PUFA) for 24 d. In vitro aorta prostacyclin (PGI2) production, serum thromboxane A2 (TxA2) production and plasma, and platelet and aorta phospholipid (PL) fatty acids were measured. Enriching the diet with n-3 PUFA resulted in significant reductions in tissue AA levels and an increase in the n-3 PUFA, particularly EPA. On this diet, the AA to EPA ratio was 1:1 in platelet PL, and it was 2:1 in the aorta PL. There were significant decreases in the in vitro PGI2 and TxA2 production compared with the control animals. The inclusion of AA in the diet resulted in marked increases in AA levels in the platelet and aorta PL with corresponding decreases in EPA. The lowest dose of AA (30 mg/rat) reversed the effects of 100 mg/day of n-3 PUFA on AA levels in platelet and aortic PL and on in vitro aorta PGI2 and serum TxA2 production. The dietary AA caused a differential (twofold) increase in TxA2 relative to PGI2 for all three levels of AA supplementation. There were greater changes in the levels of AA and/or EPA in platelet PL compared with the aorta PL, which might have accounted for the differential effects of these PUFA on thromboxane production compared with PGI2 production in this study.