We have reported that dietary fish oil (FO) leads to the incorporation of long-chain n-3 PUFA into the gut tissue of small animal models, affecting contractility, particularly of rat ileum. This study examined the FO dose response for the incorporation of n-3 PUFA into ileal tissue and how this correlated with in vitro contractility. Groups of ten to twelve 13-wk-old Wistar-Kyoto rats were fed 0, 1, 2.5, and 5% FO-supplemented diets balanced with sunflower seed oil for 4 wk, after which ileal total phospholipid FA were determined and in vitro contractility assessed. For the total phospholipid fraction, increasing the dietary FO levels led to a significant increase first evident at 1% FO, with a stepwise, nonsaturating six-fold increase in n-3 PUFA as EPA (20:5n-3), DPA (docosapentaenoic acid, 22:5n-3), and DHA, but mainly as DHA (22:6n-3), replacing the n-6 PUFA linoleic acid (18:2n-6) and arachidonic acid (20:4n-6) over the dosage range. There was no difference in KCl-induced depolarization-driven contractility. However, a significant increase in receptor-dependent maximal contractility occurred at 1% FO for carbachol and at 2.5% FO for prostaglandin E2, with a concomitant increase in sensitivity for prostaglandin E2 at 2.5 and 5% FO. These results demonstrate that significant increases in ileal membrane n-3 PUFA occurred at relatively low doses of dietary FO, with differential receptor-dependent increases in contractility observed for muscarinic and prostanoid agonists.