Although dietary fat has been associated with inflammation and cardiovascular diseases (CVD), most studies have focused on individuals with pre-existing diseases. However, the role of dietary fatty acids on inflammatory pathways prior to the onset of any abnormality may be more relevant for identifying initiating factors and interventions for CVD prevention. We fed young male pigs one of three diets differing in n-6 and n-3 polyunsaturated fatty acids (PUFA) linoleic acid (LA, 18:2n-6) and alpha linolenic acid (ALA, 18:3n-3) for 30 days. Cardiac membrane phospholipid fatty acids, phospholipase A2 (PLA₂) isoform activities, and cyclooxygenase 1 and 2 (COX-1 and 2) and 5-lipoxygenase (5-LO) expression were measured. The low PUFA diet (% energy, 1.2% LA+0.06% ALA) increased arachidonic acid (AA) and decreased eicosapentaenoic acid (EPA) in heart membranes, and increased Ca²⁺ independent iPLA₂ activity, COX-2 expression and activation of 5-LO. Increasing dietary ALA while keeping LA constant (1.4% LA+1.2% ALA) decreased the heart membrane AA and increased EPA, and prevented pro-inflammatory enzyme activation. However, regardless of high ALA, high dietary LA (11.6% LA and 1.2% ALA), decreased EPA and led to a high heart membrane AA, and Ca²⁺ dependent cPLA₂ with a marked increase in nitrosative stress. Our results suggest that the potential cardiovascular benefit of ALA is achieved only when dietary LA is reduced concomitantly, rather than fed with high LA diet. The increased nitrosative stress in the unstressed heart with high dietary LA suggests that biomarkers of nitrosative stress may offer a useful early marker of the effects of dietary fat on oxidative tissue stress.