Fatty acids of the n-3 type confer health benefits to humans and other species. Their importance to equine physiology could include improved exercise tolerance, decreased inflammation, and improved reproductive function. The circulating fatty acid profile and the acquisition and washout of fatty acids in response to n-3 supplementation were determined for horses in the current study. A fatty acid supplement high in eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid was fed to deliver EPA plus DHA at 0 (control), 10, 20, or 40 g/d to 16 mares (n = 4/group) for 28 d. Plasma was collected at -11, 3, 7, 10, 16, 23, 30, 37, 44, 70, and 87 d relative to the beginning of supplementation. Plasma was analyzed for the presence of 35 fatty acids by gas chromatography. Plasma EPA and DHA increased (P < 0.05) in a dose-responsive manner by 3 d of feeding and reached peak concentrations by 7 d. Peak EPA and DHA concentrations of the 40 g/d supplement group were approximately 13x and 10x those of controls, respectively. Plasma EPA and DHA demonstrated a steep decline (P < 0.05) from peak values by 9 d after cessation of supplementation and were near presupplementation values by 42 d. Omega-3 supplementation also increased (P < 0.05) concentrations of fatty acids C14:0, C17:1n-7, C18:1trans-11, C18:3n-6, C18:4n-3, C20:3n-6, C20:4n-6, and C22:5n-3 and decreased (P < 0.05) concentrations of C18:1cis-9 fatty acid. Seasonal effects, apparently unrelated to supplementation and likely due to the availability of fresh forage, were also noted. Unlike ruminants, there were no detectable concentrations of CLA in equine plasma. These results indicate that the circulating fatty acid milieu in horses can be influenced through targeted supplementation. Possible implications of increased n-3 plasma and tissue concentrations on specific physiological function in the equine remain to be elucidated.