Diester phthalates are industrial chemicals used primarily as plasticizers to import flexibility to polyvinylchloride plastics. In this study, we examined the hydrolysis of di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP) in human liver microsomes. These diester phthalates were hydrolyzed to monoester phthalates (mono-n-butyl phthalate (MBP) from DBP, mono-n-butyl phthalate (MBP) and monobenzyl phthalate (MBzP) from BBzP, and mono(2-ethylhexyl) phthalate (MEHP)) by human liver microsomes. DBP, BBzP and DEHP hydrolysis showed sigmoidal kinetics in V-[S] plots, and the Hill coefficient (n) ranged 1.37-1.96. The S(50), V(max) and CL(max) values for DBP hydrolysis to MBP were 99.7 μM, 17.2nmolmin(-1)mg(-1) protein and 85.6 μL min(-1)mg(-1) protein, respectively. In BBzP hydrolysis, the values of S(50) (71.7 μM), V(max) (13.0nmolmin(-1)mg(-1) protein) and CL(max) (91.3 μL min(-1)mg(-1) protein) for MBzP formation were comparable to those of DBP hydrolysis. Although the S(50) value for MBP formation was comparable to that of MBzP formation, the V(max) and CL(max) values were markedly lower (<3%) than those for MBzP formation. The S(50), V(max) and CL(max) values for DEHP hydrolysis were 8.40 μM, 0.43 nmol min(-1)mg(-1) protein and 27.5 μL min(-1)mg(-1) protein, respectively. The S(50) value was about 10% of DBP and BBzP hydrolysis, and the V(max) value was also markedly lower (<3%) than those for DBP hydrolysis and MBzP formation for BBzP hydrolysis. The ranking order of CL(max) values for monoester phthalate formation in DBP, BBzP and DEHP hydrolysis was BBzP to MBzP≥DBP to MBP>DEHP to MEHP>BBzP to MBP. These findings suggest that the hydrolysis activities of diester phthalates by human liver microsomes depend on the chemical structure, and that the metabolism profile may relate to diester phthalate toxicities, such as hormone disruption and reproductive effects.