We have investigated the effect of fish oil supplementation on the association between serum non-esterified fatty acid (NEFA) pattern and atherosclerotic activity. We studied correlations between serum non-esterified very long-chain eicosapentaenoic (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) and biochemical markers of endothelial activation before and after 18-months intervention with fish oil supplementation. The fish oil supplementation consisted of 2.4 g of EPA and DHA per day, with corn oil as placebo. Elderly men (n =171) with high risk for coronary heart disease were divided into four intervention groups in a factorial design: fish oil supplementation (n =44), dietary intervention (n =42), fish oil supplementation+dietary intervention (n =47) or placebo (n =38). The composition of fasting NEFA was analysed before and after intervention by GLC. Circulating endothelial markers were analysed by ELISA. A statistically significant positive correlation between the change in serum non-esterified DHA and soluble vascular cell adhesion molecule-1 (sVCAM-1) was found in the pooled group that received fish oil supplementation (n =91; Spearman's correlation coefficient r =0.24, P =0.02). No such correlation was found in the pooled group without fish oil supplementation (n =80). Furthermore, there was a significant negative correlation between the change in serum non-esterified EPA and the relative change in sVCAM-1 in the group that did not receive fish oil supplementation (r =-0.34, P =0.002). No such correlation was found in the group with fish oil supplementation. We conclude that large increase in serum non-esterified EPA and DHA, which can only be attained by supplementation, might increase inflammation in vascular endothelium. A moderate dietary increase in fish oil intake may, however, have an effect on decreasing inflammatory markers.