Carboxylesterases (CESs) play important roles in the metabolism of many ester-drugs. In the present study, we identified and characterized dexamethasone-induced methylprednisolone hemisuccinate (MPHS) hydrolase in rat liver microsomes. Intraperitoneal injection of dexamethasone resulted in a significant increase in the level of MPHS hydrolase activity accompanied by induction of a specific CES isozyme. Since the biochemical characteristics of the induced CES isozyme were very similar to those of rat CES RL4, we hypothesized that these were the same enzymes. The results of nano-electrospray ionization tandem mass spectrometry analysis revealed that both dexamethasone-induced CES isozyme and CES RL4 possessed identical peptide fragments to those of , a rat CES2 isozyme, supporting our hypothesis. Furthermore, the results of reverse transcription-polymerase chain reaction showed that the amount of mRNA in dexamethasone-treated liver was greater than that in control liver. To confirm that encodes dexamethasone-induced CES isozyme, cDNA cloning was performed and the obtained cDNA was expressed in Sf9 cells by using a baculovirus-mediated expression system. The recombinant CES protein could hydrolyze MPHS and exhibited biochemical characteristics similar to those of CES RL4. Collectively, the results indicated that dexamethasone-induced MPHS hydrolase in liver microsomes is a rat CES2 isozyme. Interestingly, the results also showed that this rat CES2 isozyme exists in plasma and that the amount of this protein is increased by dexamethasone. These findings, together with the findings described above, provide important information for the study of phramacokinetics and pharmacodynamics of ester-drugs as well as for the study of CESs.