Dietary polyunsaturated fatty acids (ω-3 PUFAs) are associated with reduced coronary heart disease (CHD) risk. To define mechanisms, we investigated the vasoactive properties of these fatty acids in a rat model of hypercholesterolaemia (HC) and balloon arterial injury. Mature Wistar Kyoto (WKY) rats were fed a control (C), HC-inducing diet or a HC diet after aortic balloon injury (HC+EI) for 3 weeks. Serum cholesterol (mg/dL) was increased in HC (793±31) and HC+EI (857±36) as compared to C (85±3, p≤0.001). Cumulative concentration-response curves to norepinephrine (NE, 10−9 to 10−5 mol/L) followed by docosahexaenoic (DHA) or eicosapentaenoic (EPA) (1 to 100 μmol/L) were generated in isolated aortic rings (intact and de-endothelialised, E-). Overall, contractions to NE (10−9 to 10−5 mol/L) in intact rings were diminished in HC and HC+EI groups than were apparent in the C group (p≤0.001). De-endothelialised rings exhibited much greater NE contractile responses as compared to intact rings from each of the 3 groups. Endothelium-dependent relaxations to acetylcholine (10−7 to 10−4 mol/L) were significantly decreased in the HC+EI group. Relaxant responses to the ω-3 PUFAs, DHA and EPA (1–100 μmol/L) were similar in intact rings, but enhanced in E- rings among the 3 groups. De-endothelialised ring responses were −8.6 to −35.5% (C), −8.4 to −30.5% (HC), −7.8 to −25.6% (HC+EI) to DHA (1–100 μmol/L) and −6.8 to −339% (C), −4.8 to =t-40.8% (HC), −8.9 to −34.6% (HC+EI) to EPA (1–100 μmol/L). In areas of aortic intimal hyperplasia, lipid deposition quantified spectrophotometrically using oil red O (ORO) was greater in HC+EI (0.539 μmol ORO/mm2 aortic tissue, p<0.001) as compared to C (0.292) and HC (0.315) groups. We conclude that (1) aortic contractile properties to NE are altered in rats with HC and HC+EI, (2) dietary HC after aortic injury leads to intimal hyperplasia/lipid deposition and impaired endothelium-dependent relaxations in WKY rats, and (3) ω-3 PUFAs have vasorelaxant properties in HC and HC+EI rat aorta. These properties may be beneficial in CHD.