Dietary intake of 18: 2n-6 and 18: 3n-3 may affect endogenous production and incorporation of n-3 long-chain PUFA (LCPUFA) from fish oils (FO). This double-blinded controlled 2 x 2-factorial 8-week intervention investigates the effects of high and low 18: 2n-6 intake in combination with FO-supplementation on tissue fatty acid composition. Healthy young men (n 64) were randomized to capsules with FO or olive oil (control) (4.4 (2.0-5.6) ml/d) and to either sunflower oil and margarine (S/B) or rapeseed oil and a butter spread (R/K) to provide a high or a low 18: 2n-6 intake. Diet was measured by 4-d weighed dietary records at baseline, during and 8 weeks after the intervention and tissue incorporation as fatty acid composition of peripheral blood mononuclear cells (PBMC). The fat intervention gave a mean difference in the 18: 2n-6 intake of 7.3 g/d (95 % CI 4.6, 10.0) and a similar 18: 3n-3 intake in the groups. The R/K groups had a 0.2 % fatty acid (FA%) (95 % CI 0.0, 0.4, P = 0.02) higher content of 22: 5n-3 in the PBMC, a tendency of slightly higher 20: 5n-3 (P = 0.06), but no more 22: 6n-3 (P = 0.83) than the S/B groups. FO effectively raised the PBMC content of all n-3 LCPUFA (P < 0.001). The fat intervention did not markedly influence the effect of FO; the mean PBMC content of n-3 LCPUFA was 10.3 (sem 0.3) FA% in the FO+S/B group and 10.6 (sem 0.2) FA% in the FO+R/K group. In conclusion, increasing the 18: 2n-6 intake did not have any pronounced effect on incorporation of n-3 LCPUFA in PBMC, either alone or with simultaneous FO supplementation.