Carboxylesterase (CEase) was solubilized from rat liver microsomes by autolysis followed by cholate treatment and then purified by the combination of ammonium sulfate fractionation, gel filtration, chromatography on DEAE Sephadex A-50 and hydroxyapatite and preparative Disc electrophoresis. The overall purification was 25-fold with a yield of 6% of the original enzyme activity. Analytical Disc electrophoresis of the final enzyme preparation showed a single band. However, SDS polyacrylamide gel electrophoresis revealed one main band of 93% and three other minor bands. To investigate the interaction between CEases of rat, monkey, pig and rabbit liver microsomes, rabbit antibody to the above enzyme preparation was prepared and immunological analyses, i.e., Ouchterlony's test and immunoelectrophoresis, were performed. In the comparative double diffusion test, the partial fusion of precipitation line between anti-rat CEase and the enzymes of other species was observed. In the second analysis, sharp arc precipitation lines also could be seen in all specimens and, furthermore, mobilities of each enzyme were different. These observations suggest that rat liver CEase seems to be immunologically related in part but not completely identical with the CEases of other species and the charge difference may exist in these specimens.