Fatty acid composition of phospholipids and free fatty acids were analyzed in the epithelium, stroma and endothelium of rabbit corneas. Phospholipids from endothelium had the highest proportion of arachidonate and epithelium had the lowest degree of unsaturation. Epithelium had the highest levels of free arachidonic acid. Labeled arachidonic acid was incorporated into membrane lipids of the three layers, primarily in phosphatidylcholine, phosphatidylinositol and triacylglycerols. In conclusion, the three layers of the cornea differ in the fatty acid composition of membrane lipids, particularly with regard to the polyunsaturated chains, and in the metabolism of the arachidonate chain.