Asparagus officinalis hinokiresinol synthase (HRS) is composed of two subunits, HRS alpha and HRS beta. Individually, each subunit forms (E)-hinokiresinol (EHR) from 4-coumaryl 4-coumarate, whereas a mixture of both subunits forms (Z)-hinokiresinol (ZHR) from the same substrate. In this study, we analyzed the enantiomeric compositions of ZHR and EHR formed after incubation of 4-coumaryl 4-coumarate with recombinant subunit proteins, recHRS alpha and/or recHRS beta, and with naturally occurring A. officinalis ZHR. The enantiomeric composition of ZHR formed by the mixture of recHRS alpha and recHRS beta was (+)-100% enantiomer excess (e.e.), identical to that of A. officinalis ZHR. In contrast, the enantiomeric compositions of EHR formed by recHRS alpha and recHRS beta, individually, were (-)-20.6 and (-)-9.0% e.e., respectively. These results clearly demonstrate that the subunit composition of A. officinalis HRS controls not only cis/trans isomerism but also enantioselectivity in hinokiresinol formation.