Groups of rats were fed diets containing 10% of either corn oil, partially hydrogenated soybean oil, or a mixture of the two. The partially hydrogenated oil contained a high level of trans-octadecenoate and a low level of linoleate, and all diets were adjusted to contain similar levels of cis-octadecenoate. The fatty acid compositions of five tissue lipid fractions from liver and heart (non-esterified fatty acids, acyl-CoA, diacylglycerols, triacylglycerols and phospholipids) were analyzed to measure the effect of the dietary supply on the accumulation of trans-octadecenoates and other fatty acids at different steps of glycerolipid synthesis. Although trans-octadecenoate was increased in all of the lipid fractions when the dietary supply was increased, the accumulation did not exceed 15% of the acyl chains in any of the lipid pools even when the dietary trans acid accounted for 46% of the fatty acids supplied in the diet. The trans-octadecenoate accumulated in a similar manner in the lipids of both liver and heart, and the amounts found in the acyl-CoA esters of both tissues were relatively low compared to the diet. A high dietary supply of trans-octadecenoate appeared to diminish the relative content of stearate in the acyl-CoA and phospholipid fractions. The level of cis-octadecenoate maintained in tissue phospholipids was similar to that in the acyl-CoA fractions, whereas the trans-octadecenoate content in phospholipids more closely resembled that in the diacylglycerols. Normal proportions of arachidonate were maintained in the tissue phospholipids during high intake of trans acids, even though lower levels were observed in the acyl-CoA and diacylglycerols of liver.