The effects of zero erucic acid rapeseed oil and olive oil on plasma fatty acid composition and serum cholesterol were studied in margarine users (n = 46). The replacement of margarine on bread by these oils accounted, on average, for 16 % of the total fat and 7% of the total energy intake. Fatty acid analysis of total plasma indicated a dose-dependent rise in α-linolenic (α-LLA) and oleic acid (OA) levels during rapeseed and olive oil substitutions, respectively. Rapeseed oil substitution increased the proportion of eicosapentaenoic acid (0.4 %-units, on average) in plasma phospholipids. A slight decrease in low-density lipoprotein cholesterol (LDL-C) and an increase in high-density lipoprotein cholesterol (HDL-C, 4.5%, p < 0.01) led to a significantly higher HDL-C/total cholesterol (TC) ratio (1.9 %-units). The results suggest a marked competitive effect for α-LLA, not only among plasma phospholipid fatty acids, but also in the relationships with serum lipids, since the changes in α-LLA, rather than in OA, were associated with those in LDL-C and the HDL-C/TC ratio. No competitive action of polyunsaturated acids comparable to rapeseed oil was found during olive oil substitution. In contrast to the rapeseed oil diet, the reduced proportion of linoleic acid (LA) in plasma phospholipids was not restored; this may be unfavorable if the habitual intake of LA is low. However, the effects on LDL-C levels were beneficial: the concentration decreased by 5.9% (p < 0.01), correlating inversely with the increase in OA. In addition, the concentration of HDL-C remained unchanged during olive oil substitution.