The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113–0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.