To determine the effect of dietary fish oil supplementation on myocardial dysfunction following ischemia-reperfusion, independent of plasma and blood cells, Sprague-Dawley rats were fed fish oil-rich nonpurified diet or butter-enriched diet for 5 d. Myocardial content of long-chain and (n-3) polyunsaturated fatty acids was greater in the fish oil-fed rats (P < 0.01), whereas (n-6) fatty acid content was lower compared with controls (P < 0.01). Platelet aggregation in fish oil-fed rats was also inhibited. Hearts from all rats were subjected to 15 min of global ischemia and 10 min of reperfusion. In hearts of control rats, ischemia-reperfusion resulted in a marked decrease in force of cardiac contraction, increase in coronary perfusion pressure, appearance of ventricular arrhythmias and release of creatine kinase and thromboxane B2 in the coronary effluent. Dietary fish oil supplementation attenuated myocardial dysfunction induced by ischemia-reperfusion, as indicated by smaller change in force of cardiac contraction (-77% vs. -89%, P < 0.05) and coronary perfusion pressure (+37% vs. +71%, P < 0.001). Concomitantly, release of creatine kinase as well as thromboxane B2 in coronary effluent was lower (P < 0.01). Ventricular arrhythmias occurred less frequently in hearts from fish oil-fed rats. Thus, short-term dietary fish oil supplementation attenuates myocardial dysfunction caused by ischemia-reperfusion by a direct effect on the heart independent of blood and plasma components.