Fifteen forms of carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of rats, mouse, hamster, guinea pig, rabbit, beagle dog, pig, cow, crab eating monkey and humans by the same procedure used, and their physical, enzymological and immunological properties were compared with each others. The substrate specificity and immunological reactivity of liver microsomal carboxylesterase from above animals were also examined for comparison. The fifteen purified preparations have similar subunit weight (57, 000-64, 000), but their isoelectric point differ widely (4.7-6.5). The purification procedure of all isozymes include concanavalin A-Sepharose column chromatography. The isozymes were not eluted from the column with a high concentration of sodium chloride, but were efficiently eluted with alpha-methylmannoside. This observation suggested that the carboxylesterase studied are glycoproteins. All the isozymes except rat RL1and RL2 possess a high hydrolytic activity toward all the substrates examined. Long-chain monoacylglycerol was hydrolyzed by the purified carboxylesterase isozymes except rat RL1 and cow B1. Anti-rat RH1 IgG was found to possess high cross-reactivity with all isozymes tested, except monkey MK2, by immunoblotting analysis. The amino acid composition of carboxylesterase isozymes showed considerable similarities, except for monkey MK2. The amino-terminal amino acid sequences showed a striking homology, except for monkey MK2, though the first amino acid in the sequence was different in every isozymes. On the other hand, the well-known peroxisome proliferators, such as clofibrate, diethylhexylphtalate, and perfluorinated fatty acid treatment extraordinary induced the carboxylesterases in rats and mice, and slightly induced in hamsters. Whereas, guinea pig liver carboxylesterase was not induced. Hepatic microsomal carboxylesterases in mammals play an important role in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in physical, enzymatic, and immunochemical properties, but not similar in induction of peroxisome proliferators.