The noncompetitive and-competitive hydrolyses of (2R, 4’R, 8’R)-alpha-tocopheryl acetate (RRR-alpha-TOAc, the acetate of natural vitamin E) and (2S,4’R,8’R)-alpha-tocopheryl acetate (SRR-alpha-TOAc) catalyzed by crude and pure bovine cholesterol esterase (BCE) and crude and pure porcine cholesterol esterase (PCE) have been studied at 37 degrees C. These two CE’s are catalytically active toward tocopheryl acetates only in the presence of bile salts. The three 3 alpha,7 alpha,12 alpha-trihydroxy bile salts, cholate, glycocholate and taurocholate, not only are effective activators of BCE and PCE, but also modulate the diastereoselectivities of these two enzymes in their hydrolyses of RRR- and SRR-alpha-TOAc in a characteristic manner. Rates of hydrolyses were much faster in the presence of a small quantity of dl- or l-dimyristoylphosphatidylcholine (DMPC) than in its absence. However, for each enzyme, the direction and even the magnitude of the diastereoselectivity is primarily determined by the bile salt employed and not by the presence or nature of the co-lipid (DMPC or sodium oleate), nor by the bile salt/co-lipid ratio, nor by the purity of the enzyme. In noncompetitive experiments, the ratios of the BCE-catalyzed initial rates of hydrolyses of the diastereomeric acetates, V-i(RRR)/V-i(SRR), are 0.21, 1.5, and 2.7 for cholate, glycocholate, and taurocholate, respectively, and for the PCE-catalyzed noncompetitive reactions, 0.21, 7,9, and 7.5 for the same three bile salts. In competitive experiments cholate, respectively.