Background Many health effects of oils rich in oleic acid (18:1 n9) seem to be opposite those of arachidonic acid (20:4 n6), i.e. concerning cardiovascular risk. In recent study in rats we observed that percentages of oleic acid and arachidonic acid were inversely related in total serum lipids. In the present work we investigate whether an inverse relationship between this couple of fatty acids also exists in the phospholipid fraction of human sera. Methods The study group consisted of 11 men and 35 women. Mean age was 23.8 ± 2.5 years (mean ± SD), and the body mass index was 23.5 ± 3.2 kg/m2. After fasting overnight, blood was drawn and the concentration of fatty acids in serum phospholipids was determined, using gas chromatography. We studied the association between percentages of oleic acid and arachidonic acid using bivariate correlations (Pearson), and multiple linear regressions. Results We found an inverse relationship (r = −0.563, p < 0.001; n = 46) between % oleic acid and % arachidonic acid in the serum phosholipid fraction of the 46 fasting subjects. By multiple linear regression, and % 20:4 n6 as the dependent variable, the inverse association with % 18:1 n9 persisted when controlling for sex, age, body mass index, and percentages of the other fatty acids measured (t = −17.6, p < 0.001). Per cent 20:4 n6 seemed to correlate negatively (r = −0.289, p = 0.05) with the (18:1 n9)/(18:0) ratio, estimating Delta9 desaturase activity, and % oleic acid correlated negatively (r = −0.321, p = 0.029) with the (20:4)/(18:2) ratio, estimating desaturases/elongase activities. Conclusions In a group of healthy human subjects, percentages of oleic acid and arachidonic acid were inversely related, and the inverse association persisted when controlling for possible confounding variables. The findings might contribute to explain positive health effects of foods rich in oleic acid.