A major function of the enterocytes is absorption, processing, and export of dietary lipids into the lymphatic system. Pluronic L-81 is a non-ionic hydrophobic surfactant, which specifically inhibits lipid absorption in the intestine when administered in vivo. This compound is therefore an attractive probe to perturb and investigate the molecular and intracellular events in chylomicron assembly in the enterocytes. In the present study pluronic acid was administered to hamsters followed by isolation of the enterocytes and cell fractionation to investigate the effect of pluronic acid on intracellular events in lipid absorption. Four types of diet were administered to hamsters for three weeks; low-fat chow, high-fat chow and each diet with or without added pluronic acid. Sub-cellular fractions of freshly isolated enterocytes were prepared. Consistent with morphological observation, the high fat diet resulted in a three-fold increase in the triacylglycerol (TAG) content of the enterocytes and addition of pluronic acid to either the low fat or the high fat diets resulted in a ten-fold increase in cellular TAG levels. Determination of the mass of TAG and the time course of incorporation of 3H-triolein, administered by gavage, showed that the increased TAG was recovered in the microsomal (endoplasmic reticulum) fraction and the cytosol. In microsomes, increased TAG was recovered mainly in the membrane although there was a significant amount in the microsomal luminal contents. Pluronic acid therefore acts at the level of the endoplasmic reticulum and inhibits the assembly of apo-B48 with chylomicron TAG. The excess TAG is transferred to cytosolic stores.