Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10-12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S-adenosyl-[methyl-3H]methionine resulted in the formation of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N]dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The Km for S-adenosylmethionine was 1.10(-4) M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4-5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na+-dependent uptake of either D-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.