AbstractRats fed a fat‐free diet from weaning were contined on that diet alone or supplemented with methyl linoleate, methyl linoleate plus a mixture of antibiotics, or methyl arachidonate.Dietary linoleate and arachidonate reduced the concentration of octadecenoic acid and increased that of stearic acid in the mucosa and luminal lipids. This effect was prevented in the mucosa but not in the intestinal contents by antibiotic supplementation of the linoleate diet.Evidence for the conversion of linoleic into eicosatetraenoic acid was found in both mucosa and luminal lipids. The conversion was impaired by the addition of antibiotics to the diet.Linoleate feeding combined with antibiotic addition provided evidence for the intestinal hydrogenation of dietary linoleic into either octadecenoic or stearic acids by separate routes, the latter being impaired by antibiotic ingestion.The ingestion of methyl linoleate or arachidonate modified only slightly the fecal fatty acid pattern of rats previously on a fat‐free diet.