Unlike most esterases, which are predominantly bound to the microsomal fraction, the enzymes hydrolyzing acetylsalicylic acid are present in an equal amount in the cytosol. Two soluble isozymes were purified to homogeneity from rat liver and characterized as serine esterases with a Mr of 35,000. Both had the wide substrate spectrum characteristic of enzymes active in detoxication. Both had a very low Km for acetylsalicylate. Three other cytoplasmic enzymes active with aspirin were observed but these differed in their high Mr (about 220,000) and their lack of reactivity with antibody to one of the homogeneous isozymes.