An acetanilide-hydrolyzing esterase of rat liver microsomes was found to be easily solubilized from the membranes by treatment with phospholipase A [EC 3. 1. 1. 4] (pH 6.8), aqueous acetone, low concentrations of detergents, alkali, or sonic oscillation. Most microsomal enzymes, such as cytochrome b5 and NADPH-cytochrome c reductase, were not solubilized by these treatments. On the other hand, digestion with trypsin [EC 3. 4. 4. 4] did not liberate the esterase from the membranes, although it solubilized cytochrome b5 and NADPH-cytochrome c reductase very effectively. The esterase was extracted from an acetone powder of trypsin-treated rat liver microsomes and purified about 50-fold by Sephadex G-150 gel filtration and DEAE-Sephadex chromatography. Rabbit antiserum was prepared against the purified esterase. The antibody thus prepared did not inhibit the acetanilide-hydrolyzing activity of the purified enzyme, but precipitated this activity efficiently. However, evidence was obtained that the antibody did not react with the esterase in untreated microsomal vesicles. This suggests that the enzyme in microsomes is not located on the outer surface of the vesicular membranes. These results are discussed in relation to the binding of the esterase to the endoplasmic reticulum.