Phospholipid analogs in which the acyl-oxyester bond is replaced by an acyl-thioester bond represent convenient substrates for sensitive assays of lipolytic enzymes. It has previously been found that such thioester substrates are hydrolyzed at higher rates than their oxyester counterparts. For bovine liver lysophospholipase II the preferential hydrolysis of thioesters appeared to be due to the thioester linkage per se rather than to the formation of preferred interfaces. The preferential hydrolysis of thioesters persisted when thioester and oxyester substrates were presented to the enzyme either as mixed micelles or incorporated in the bilayer of phospholipid vesicles. The transbilayer distribution of thioester and oxyester substrates is sonicated phospholipid vesicles is identical with no apparent indications for transbilayer movement of both substrates.