Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.