The effect of chronic feeding (3-4 wk) of the hydrophobic surfactant, Pluronic L-81, on the lymphatic transport of triglyceride and cholesterol was studied in rats with thoracic duct fistula. A lipid emulsion containing [3H]triolein (13.3 mM), [14C]cholesterol (2.6 mM), phosphatidylcholine (2.9 mM), sodium taurocholate (19 mM), with 0.17 mg/ml (experimental) or without Pluronic L-81 (L-81) added (control) was infused at the rate of 3 ml/h. Lymph triglyceride and cholesterol outputs were greatly impaired in the experimental rats compared to the control rats. The phospholipid output compared to the control was also reduced but to a lesser extent in the experimental rats. Comparable recovery of radioactive 3H-labeled lipid and [14C]cholesterol from the intestinal lumen of control and experimental rats showed that digestion and absorption were not impaired in the experimental rats. The distribution of mucosal 3H radioactivity in various lipid fractions showed no impairment in reesterification. The greatly depressed lymphatic lipid transport was associated with marked accumulation of absorbed lipid in enterocytes, suggesting that Pluronic L-81 interferes with lipoprotein assembly and/or exit of lipoproteins from the mucosal cells. The animals fed chronically for 4-6 wk regained their ability to transport lipid 24 h after termination of L-81 feeding. The effect of this agent, therefore, is readily reversible.