In response to herbivory, poplar produces among other compounds the volatile alcohol 2-phenylethanol and its corresponding glucoside 2-phenylethyl-β-D-glucopyranoside. While the free alcohol is released only upon herbivory, the glucoside accumulates also in undamaged leaves, but increases after herbivore feeding. Recently we showed that 2-phenylethanol and its glucoside are biosynthesized via separate pathways in Populus trichocarpa. The phenylacetaldehyde synthase PtAAS1 plays a central role in the de novo formation of herbivory-induced volatile 2-phenylethanol, while the phenylalanine decarboxylase PtAADC1 initiates a pathway responsible for the herbivory-induced production of 2-phenylethyl-β-D-glucopyranoside. Besides PtAAS1, P. trichocarpa possesses another aromatic aldehyde synthase PtAAS2 with in vitro enzymatic activity comparable to that of PtAAS1. However, in contrast to PtAAS1, which is exclusively expressed in herbivory-damaged leaves, PtAAS2 was found to be expressed at constant levels in both damaged and undamaged leaves. Thus it has been hypothesized that PtAAS2 provides phenylacetaldehyde as substrate for the constitutive formation of 2-phenylethyl-β-D-glucopyranoside in undamaged P. trichocarpa trees. By generating RNAi-mediated AAS2 knockdown plants, we show here that despite the similar activities of PtAAS1 and PtAAS2 in vitro, the latter enzyme does not contribute to the biosynthesis of 2-phenylethyl-β-D-glucopyranoside in planta. Based on the recent finding that phenylpyruvic acid accumulates in undamaged poplar leaves, the constitutive formation of the glucoside may now be suggested to proceed via the Ehrlich pathway, which begins with the conversion of phenylalanine into phenylpyruvic acid.