Induction of adventitious roots (ARs) is an auxin-regulated developmental process which is negatively affected by the maturity of the plant. Analysis of the sequence encoded by the CsGH3-1 gene isolated from chestnut revealed that the putative protein belongs to group II of the GH3 family and contains the enzyme domains involved in conjugating indole-3-acetic acid (IAA) to amino acids. Here, we characterized the gene expression induced in chestnut microshoots treated with indole-3 butyric acid (IBA) to promote AR formation. Expression of CsGH3-1 was strongly induced in IBA-treated microshoots, whereas the transcripts were barely detected in control (untreated) microshoots. Comparison of the expression patterns in the rooting-competent and rooting-incompetent shoots confirmed that expression of the gene was correlated with the ontogenetic stage of the microshoots. The CsGH3-1 mRNA levels were higher in mature shoots than in rooting-competent shoots during the period of cell dedifferentiation prior to cell division, which suggests a putative role for the gene in this process. Furthermore, accumulation of the transcripts in the cambium cells, only detected in root-forming shoots, was highly correlated with the ability of these cells to generate ARs. Overall, our findings indicate that the CsGH3-1 gene is involved in auxin homeostasis and has a tissuespecific function in generating the auxin gradient required for transition of cambium cells into root initials during induction of ARs.