Triacylglycerols (TAG) are the primary sources of preformed fatty acids (FA) for lipid synthesis in the mammary gland. However, polyunsaturated FA escaping ruminal biohydrogenation are selectively incorporated into cholesterol esters (CE) and phospholipids (PL). The aim of the current experiment was to study the effects of abomasal infusion of increasing amount of linseed oil (L-oil) on plasma distribution of α-linolenic acid (α-LA) and its transfer efficiency into milk fat. Five rumen-fistulated Holstein cows were randomly distributed in a 5 × 5 Latin square design. Abomasal infusion of L-oil (55.9% α-LA) was performed at the rate of 0, 75, 150, 300, and 600 ml/d. Concentrations of α-LA increased quadratically in TAG, PL, and CE; a less steep slope was observed with an inflexion at an infusion rate of 300 ml L-oil per day. The increase in plasma concentration of α-LA was of a lower magnitude in CE as compared with the other two fractions, resulting in a quadratic decrease in relative proportion of this FA circulating as CE. The transfer efficiency into milk fat increased from 0 to 150 ml L-oil infused, and a plateau was maintained thereafter with greater levels of infusion (quadratic response). This pattern resembles the quadratic response of the relative proportion of α-LA circulating as TAG, and the relative concentration of this FA in TAG. Increasing the postruminal supply of α-LA partly overcame the segregation mechanism of absorbed polyunsaturated FA in different plasma lipid classes. Proportionately more α-LA was then esterified as TAG, at the expense of CE, increasing its efficiency of transfer into milk fat. This mechanism appears to be surpassed in its turn when L-oil infusion was increased over 150 ml/d. Nevertheless, the yield of α-LA in milk fat continued to increase, but at a slower rate at the highest levels of infusion.