Differences in esterase expression among human, rhesus monkey, cynomolgus monkey, dog, minipig, rabbit, rat, and mouse plasma were identified using native polyacrylamide gel electrophoresis. Paraoxonase (PON) and butyrylcholinesterase (BChE) were ubiquitous in all species, but were highly expressed in primates and dogs, whereas carboxylesterase (CES) was only abundant in rabbits, mice, and rats. Several unknown esterases were observed in minipig and mouse plasma. These differences in plasma esterases and their expression levels result in species differences with respect to hydrolase activity. These differences were characterized using several different substrates. In contrast to the high hydrolase activity found for p-nitrophenylacetate (PNPA), a substrate of several hydrolase enzymes, irinotecan, a carbamate compound, was resistant to all plasma esterases. Oseltamivir, temocapril, and propranolol (PL) derivatives were rapidly hydrolyzed in mouse and rat plasma by their highly active CES enzyme, but rabbit plasma CES hydrolyzed only the PL derivatives. Interestingly, PL derivatives were highly hydrolyzed by monkey plasma BChE, whereas BChE from human, dog, and minipig plasma showed negligible activity. In conclusion, the esterase expression and hydrolyzing pattern of dog plasma were found to be closest to that of human plasma. These differences should be considered when selecting model animals for preclinical studies. (C) 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:39793988, 2012