Previous studies have not addressed the effect of differing fat intake on the effectiveness of varying (n-3) polyunsaturated fatty acid (PUFA) ingestion in altering tissue composition and eicosanoid production. This study examined (n-3):(n-6) PUFA ratios of 0, 0.1:1, 0.2:1, 0.4:1, and 1:1 with total fat at 5, 10, 15, and 20 g/100 g of diet and (n-6) PUFA fixed at 1.5 g/100 g of diet on tissue composition and peritoneal cell eicosanoid response to an in vivo inflammatory stimulus in 240 mice. Both (n-3) PUFA and total fat intake influenced tissue composition and eicosanoid biosynthesis. Increased (n-3) PUFA intake was associated with an increase in tissue (n-3) PUFA and a decrease in long-chain (n-6) PUFA. Although hepatic tissue linoleic acid (LA) was not altered by (n-3) PUFA intake or changes in total fat, peritoneal cell LA increased in response to increasing total fat but was unaffected by changes in dietary (n-3) PUFA. Four-series leukotrienes (LT) decreased progressively with increased (n-3) PUFA at all fat intake levels. In addition, four-series LT decreased with increased total fat at low (n-3):(n-6) ratios (0 and 0.1). At high (n-3):(n-6) ratios (0.4 and 1.0) increasing dietary fat between the 5 and 15 g/100 g diets increased four-series LT synthesis, which reached a plateau between 15 and 20 g fat/100 g diets. Five-series LT production generally rose with increased (n-3) PUFA intake; this effect was most evident in mice fed the 5 g fat/100 g diet. Increasing total dietary fat at the three highest (n-3):(n-6) ratios (0.2, 0.4, 1.0) decreased five-series LT production. Elevated (n-3) PUFA and total fat intake exerted an additive effect with respect to prostacyclin (PGI(2)) production because it was reduced with increasing intakes of both. Compared with the mice consuming the no (n-3) 5 g/100 g diets, PGI(2) levels were reduced by 88% in mice consuming the highest total fat and (n-3) PUFA diets. At low fat intake (5 and 10 g/100 g diet), increasing the (n-3) PUFA intake was associated with a decrease in PGE(2) synthesis. However, unlike PGI(2), high fat intake reduced PGE(2) to basal levels with no further reduction induced by increased (n-3) PUFA intake.