We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control). The responsiveness to ouabain was evaluated for the two groups in Langendorff-perfused hearts, by ³¹P nuclear magnetic resonance spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum positive inotropy achieved with ouabain was nearly two times higher in the FO than in the control group and was not associated with significant changes in energetics. Alteration of function and energetic metabolism and inhibition of Na-K-ATPase in response to 3 × 10⁴ M ouabain were delayed in the FO group. This study demonstrates that dietary FO, by a cardiac membrane incorporation of n-3 polyunsaturated fatty acid, promotes positive inotropy of ouabain without toxicity and changes in cardiac metabolism.