[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.