Rats were fed semipurified diets containing 10% fat with a constant polyunsaturated/monounsaturated/saturated fatty acid ratio of 1:1:1. This was performed by mixing vegetable oils. Linoleic acid was the sole polyunsaturated fatty acid in the linoleic acid group. The alpha-linolenic and arachidonic acids were contained at the 1% level in the diet at the expense of linoleic acid in the alpha-linolenic acid and arachidonic acid groups, respectively. The concentration of serum triglyceride, phospholipid and liver triglyceride were significantly lower in the alpha-linolenic acid and arachidonic acid groups than in the linoleic acid group. Hepatic phospholipids were significantly higher in the alpha-linolenic acid and arachidonic acid group as compared to the linoleic acid group. Compared to the linoleic acid group, the percentages of arachidonic acid in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the liver and heart were significantly lower in the alpha-linolenic acid group; in contrast, they were significantly higher in the arachidonic acid group. Dietary arachidonic acid, but not alpha-linolenic acid, resulted in a marked reduction of linoleic acid and increased arachidonic and n-6 docosapentaenoic acids in heart cardiolipin. Platelet thromboxane (TX) A2 production tended to be lower in the alpha-linolenic acid group as compared to the linoleic acid and arachidonic acid groups. There was no difference in TXA2 production between the linoleic acid and arachidonic acid groups. The aortic production of prostacyclin (PGI2) was the same among the three groups. The ratios of TXA2 and PGI2 in the linoleic acid and arachidonic acid groups were comparable, whereas they were significantly higher than that in the alpha-linolenic acid group. The results demonstrate that dietary arachidonic acid enriches the arachidonic acid content in tissue phospholipids more effectively than linoleic acid, whereas the effect on the production of TXA2 and PGI2 was comparable under these experimental conditions.