In vitro studies were performed to investigate a possible mechanism by which lecithin suppresses intestinal cholesterol absorption. The hypothesis that lecithin acts by retarding the diffusion of micelles across the unstirred water layer (UWL) was tested by measuring cholesterol influxes (Ji) in segments of rat intestine under conditions with no maximal and minimal effective thickness of the UWL. Ji was sharply reduced by lecithin under all conditions with no apparent relationship to the thickness of the UWL. The hypothesis was further tested by determining if lecithin had any effect on the molecular weight of the micelles. Gel filtration studies indicated no change in molecular weight (range, 21,000-24,000). Finally, actual measurements of the coefficients of free diffusion (D) of the micelles revealed that lecithin in the concentration that caused a 42-90% reduction in Ji caused only a 22% reduction in D. The basis for the decrease in D is unknown, but it was sufficient to account for the decrease in Ji. Therefore, except for the small decrease in micellar diffusion coefficient as a possible contributing factor, the data offered little support for the hypothesis and we concluded that lecithin suppresses cholesterol absorption by some other or additional mechanism, such as by a direct effect on cell membrane or by holding the cholesterol absorption by some other or additional mechanism, such as by direct effect on the cell membrane or by holding the cholesterol in micellar form to reduce its partition coefficient.