Diester phthalates are industrial chemicals used primarily as plasticisers to import flexibility to polyvinylchloride plastics. In this study, the authors examined the hydrolysis of di-Bu phthalate (DBP), butylbenzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP) in human liver microsomes. These diester phthalates were hydrolysed to monoester phthalates (mono-Bu phthalate (MBP) from DBP, mono-Bu 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-[fusion_builder_container hundred_percent=”yes” overflow=”visible”][fusion_builder_row][fusion_builder_column type=”1_1″ background_position=”left top” background_color=”” border_size=”” border_color=”” border_style=”solid” spacing=”yes” background_image=”” background_repeat=”no-repeat” padding=”” margin_top=”0px” margin_bottom=”0px” class=”” id=”” animation_type=”” animation_speed=”0.3″ animation_direction=”left” hide_on_mobile=”no” center_content=”no” min_height=”none”][S] plots, and the Hill coefficient (n) ranged 1.37-1.96. The S50, Vmax and CLmax values for DBP hydrolysis to MBP were 99.7 íM, 17.2 nmol/min/mg protein and 85.6 íL/min/mg protein, respectively. In BBzP hydrolysis, the values of S50 (71.7 íM), Vmax (13.0 nmol/min/ mg protein) and CLmax (91.3 íL min-1 mg-1 protein) for MBzP formation were comparable to those of DBP hydrolysis. Although the S50 value for MBP formation was comparable to that of MBzP formation, the Vmax and CLmax values were markedly lower (<3%) than those for MBzP formation. The S50, Vmax and CLmax values for DEHP hydrolysis were 8.40 íM, 0.43 nmol/min/mg protein and 27.5 íL/min/mg protein, respectively. The S50 value was about 10% of DBP and BBzP hydrolysis, and the Vmax value was also markedly lower (<3%) than those for DBP hydrolysis and MBzP formation for BBzP hydrolysis. The ranking order of CLmax values for monoester phthalate formation in DBP, BBzP and DEHP hydrolysis was BBzP to MBzP g 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 metabolyte profile may relate to diester phthalate toxicities, such as hormone disruption and reproductive effects.