Background/Aim: N-6 fatty acids are considered to promote diseases prevalent in industrialized countries and characterized by an increased eicosanoid biosynthesis from arachidonic acid (AA). We investigated the impact of the linoleic acid (LA) intake on AA levels in humans. Methods: Six healthy female volunteers (age range 23–34 years) were given liquid formula diets (LFD) devoid of AA for 6 weeks, providing a constant intake of zero energy% (LFD 0: protein 15%, carbohydrates 85%) or 20 energy% (LFD 20: protein 15%, carbohydrates 55%, fat 30%) LA, for 3 weeks each. Fatty acids of plasma cholesteryl esters and platelet lipids were determined each week, and the prostaglandin biosynthesis was measured in 24-hour urine samples. Results: LFD 0 increased (+31% of initial value) and LFD 20 lowered (–30% of initial value) the percentage of AA in plasma cholesteryl esters and platelet lipids. Moreover, absence of dietary AA lowered the percentages of AA in plasma (–31% week 0 vs. week 6) and platelet (–11%) lipids, indicating a low transformation of LA. LFD 0 reduced urinary metabolite levels of prostaglandins D, E, and F in 24-hour urine samples (–48%, p < 0.001) within 24 h, but did not significantly affect platelet aggregation (–10%) and thromboxane formation (–25%). LFD 20 significantly lowered platelet aggregation (–25%) and thromboxane formation (–43%). The prostaglandin metabolite levels increased during the first 10 days, declined thereafter, and were lower than the preexperimental values at the end of the 3-week period. Conclusions: The results show that dietary LA does not increase the AA levels in plasma or platelet lipids and does not persistently contribute to prostaglandin biosynthesis which is increased by AA intake with Western diets.