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Single bilayer vesicles were prepared from total rat liver microsomal lipids to which 5 mol% lysophosphatidylcholine had been added. The availability of lysophosphatidylcholine for enzymatic hydrolysis by lysophospholipase (EC 126.96.36.199) was found to be higher in vesicles prepared by the cholate dispersion technique when compared with sonicated vesicles. Sepharose 4 B chromatography showed that the vesicles prepared by the cholate technique were smaller than those prepared by sonication. This is in contrast to previous observations for egg phosphatidylcholine vesicles. Total rat liver microsomal extracts were found to contain proteolipid, which could be removed by ether precipitation. Cholate vesicles prepared from proteolipid-free extracts were still smaller than sonicated vesicles from this extract. Experiments with [14C] dextran entrapped in the vesicles indicate that there is no loss of the permeability barrier of the vesicles for high molecular weight solutes during vesicle treatment with lysophospholipase. The high availability of lysophosphatidylcholine in cholate vesicles of total rat liver microsomal lipids is discussed in terms of a highly asymmetric distribution of lysophosphatidylcholine over the inner and outer monolayer of the bilayer. 350c69d7ab