Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil may prevent development of heart failure through alterations in cardiac phospholipids that favorably impact inflammation and energy metabolism. A high-fat diet may block these effects in chronically stressed myocardium. Pathological left ventricle (LV) hypertrophy was generated by subjecting rats to pressure overload by constriction of the abdominal aorta. Animals were fed: (1) standard diet (10% of energy from fat), (2) standard diet with EPA+DHA (2.3% of energy intake as EPA+DHA), (3) high fat (60% fat); or (4) high fat with EPA+DHA. Pressure overload increased LV mass by approximately 40% in both standard and high-fat diets without fish oil. Supplementation with fish oil increased their incorporation into cardiac phospholipids, and decreased the proinflammatory fatty acid arachidonic acid and urine thromboxane B(2) with both the standard and high-fat diet. Linoleic acid and tetralinoloyl cardiolipin (an essential mitochondrial phospholipid) were decreased with pressure overload on standard diet, which was prevented by fish oil. Animals fed high-fat diet had decreased linoleic acid and tetralinoloyl cardiolipin regardless of fish oil supplementation. Fish oil limited LV hypertrophy on the standard diet, and prevented upregulation of fetal genes associated with heart failure (myosin heavy chain-beta and atrial natriuetic factor). These beneficial effects of fish oil were absent in animals on the high-fat diet. In conclusion, whereas treatment with EPA+DHA prevented tetralinoloyl cardiolipin depletion, LV hypertrophy, and abnormal genes expression with pressure overload, these effects were absent with a high-fat diet.