Technical Abstract: Indole-3-acetic acid (IAA) is the most active endogenous auxin involved in various physiological processes in higher plants. Concentrations of IAA in plant tissues are regulated at multiple levels including de novo biosynthesis, degradation, and conjugation/deconjugation. In this paper, we report identification and biochemical characterization of a gene from poplar (Populus trichocarpa) encoding IAA methyltransferase (IAMT) that also plays a role in regulating homeostasis of IAA. Poplar IAMT (PtIAMT1) was identified from the poplar genome based on its sequence relatedness to Arabidopsis IAMT. A full-length cDNA of PtIAMT1 was cloned from poplar leaves via RT-PCR. Recombinant PtIAMT1 expressed in E. coli was purified to electrophoresis homogeneity. Enzyme assays combined with GC-MS verified that PtIAMT1 catalyzes the formation of methyl indole-3-acetate using S-adenosyl-L-methionine as a methyl donor and IAA as a methyl acceptor. In addition to using IAA as substrate, PtIAMT1 showed activity, although at a lower level, with another endogenous auxin, indole-3-butyric acid. PtIAMT1 has a pH optimum at pH7.5. Its activity can be promoted by K+ but inhibited by Fe2+, Cu2+ and Zn2+. Under steady-state conditions, PtIAMT1 exhibited Km values of 23 'M and 30 'M for IAA and SAM, respectively. Gene expression analysis showed that PtIAMT1 has the highest level of expression in stems, a moderate level of expression in young leaves, and a low level of expression in roots. The presence of PtIAMT1 transcripts in several organs suggests that PtIAMT1 is involved in multiple biological processes in poplar.