Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 μM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC(50) values of 0.51 and 0.36 μM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.