We investigated the ester formation of gypsophila (Gypsophila paniculata L. 'Bristol Fairy') inflorescences by exogenously applying several alcohols and determining the substrate specificity of the esterification enzyme, alcohol acetyltransferase (AAT). All exogenous alcohols tested except for ethanol were converted to the respective acetate esters in gypsophila flowers, which indicated that exogenous alcohols, catalyzed by AAT, could react with endogenous acetyl-CoA. Application of isoamyl alcohol facilitated the production of isoamyl isovalerate and consequently decreased emission of methylbutyric acids, which is responsible for the unpleasant odor of gypsophila inflorescences. Some aromatic alcohols, i.e., benzyl alcohol and 2-phenylethyl alcohol, also had similar effects. cis-3-Hexene 1-ol and 1-hexanol were the most reactive alcohol substrates catalyzed by AAT in the cell-free extract, whereas ethanol was the least reactive, indicating that the substrate specificity of AAT in vitro is correlated with that in vivo. That AAT activity in cell-free extracts was already prominent at the bud stage, but decreased when the florets opened suggests that the scarcity of alcoholic substrates is a major factor in limiting volatile ester emission from gypsophila inflorescences.