Lymph fistula rats were used to determine the acute effects of the hydrophobic surfactant, Pluronic L-81, on lipid transport by the small bowel. Animals were infused intraduodenally with a lipid emulsion containing [3H]triolein and Pluronic L-81, and the rate of intestinal transport of absorbed lipid into lymph was studied using liquid scintillation spectrometry. With this technique, various dose levels of Pluronic L-81 were analyzed for a possible inhibitory effect on lipid transport. Also, the rate at which this agent produced inhibition of intestinal lipid transport was determined. Results were correlated with electron microscopic studies of jejunal enterocytes and lipoprotein particles recovered in intestinal lymph. Infusion of Pluronic L-81 at a rate of 0.25 mg/h had no effect, but infusion at 0.5 and 1 mg/h produced a dramatic reduction in lipid transport by the small bowel. The rate of inhibition of lymphatic lipid output was rapid, with a t1/2 of 69 min for the 0.5 mg/h dose and 35 min for the 1 mg/h dose. This inhibition of lipid transport was associated with marked mucosal accumulation of lipid as demonstrated by radiochemical and morphological data. By electron microscopic analysis, only very low-density lipoprotein-sized particles were transported into lymph by enterocytes exposed to an effective dose of Pluronic L-81. It is concluded that small amounts of Pluronic L-81 produce a striking inhibition in the intracellular transport of chylomicron-sized particles, thereby blocking secretion of chylomicrons by the enterocytes. Furthermore, this action is very rapidly produced by effective doses of this agent.