The effect of fish oil supplementation on the nonesterified fatty acid (NEFA) concentration and composition in the normoxic and hypoxic myocardium of pigs was examined. Two groups of female pigs (n = 7) were fed a diet supplemented with either 5 g beef tallow/kg (as control) or 5 g fish oil/kg (MaxEPA) rich in (n-3) fatty acids. After 6 wk of supplementation, the pigs were anesthetized, hearts exposed by thoracotomy followed by occlusion of the left anterior descending artery. Normoxic and hypoxic regions of the heart were examined for NEFA concentration and composition by using a combination of thin layer and gas chromatography. Nonesterified (n-6) and (n-3) fatty acid concentration and composition differed significantly between the two groups in both the normoxic and hypoxic areas of the heart. Eicosapentaenoic and docosahexaenoic acid concentration in the NEFA fraction of the normoxic myocardium were higher in the fish oil group than in the beef tallow group (P < 0.001). In the fish oil-fed pigs, the (n-3) NEFA concentration was significantly higher in the hypoxic compared to the normoxic region of the heart. The fish oil-fed group had lower levels of arachidonic acid in the NEFA fraction compared to the beef tallow-fed group, whereas the hypoxic myocardium had higher levels of arachidonic acid, regardless of the dietary fat supplementation. Despite large differences in the proportions of saturated fatty acids in the experimental diets, there was little or no difference in the saturated fatty acid content of cardiac phospholipid and NEFA fractions. Following myocardial ischemia, (n-3) fatty acids in the NEFA fractions were selectively increased in the fish oil-fed pigs, implicating the possible role of nonesterified (n-3) polyunsaturated fatty acids in the prevention of arrhythmias.