Properties of the intestinal digestion of the dietary phytosterols, cholesterol and cholestanol, and the mechanisms by which phytosterols inhibit the intestinal absorption of cholesterol in healthy human subjects are poorly known. We have studied the hydrolysis of dietary plant sterol and stanol esters and their subsequent micellar solubilization by determining their concentrations in micellar and oil phases of the jejunal contents. Two liquid formulas with low (formula 1) and high (formula 2) plant stanol concentrations were infused via a nasogastric tube to the descending duodenum of 8 healthy human subjects, and intestinal contents were sampled for gas-liquid chromatographic sterol analysis 60 cm more distally. During the duodenal transit, phytosterol esters were hydrolyzed. This was especially profound for sitostanol, as its esterified fraction per milligram of sitosterol decreased 80% (P < 0.001) in formula 1 and 61% (P < 0.001) in formula 2. Contrary to that, esterified fraction of cholesterol per milligram of sitosterol was increased fourfold (P < 0.001) in formula 1 and almost sixfold (P < 0.001) in formula 2, whereas that of cholestanol remained unchanged. Percentages of esterified sterols and stanols in total intestinal fluid samples were higher after the administration of formula 2 than of formula 1. Esterified cholesterol and stanols accumulated in the oil phase, and free stanols replaced cholesterol in the micellar phase. At high intestinal plant stanol concentrations, cholesterol looses its micellar solubility possibly by replacement of its free fraction in the micellar phase by hydrolyzed plant stanols, which leads to a decreased intestinal absorption of cholesterol.