Cotyledon explants of walnut (Juglans regia) have been shown to generate adventitious roots on growth regulator-free medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) and its dynamic changes during adventitious root formation were investigated using an in situ immunohistochemical approach. Before root induction, IAA signal was distributed throughout the freshly excised cotyledon explants. During provascular bundle differentiation, the IAA signal was mainly located in the provascular bundles. At the stage of annular meristematic zones formation, the IAA signal was mainly distributed in the meristematic zones and decreased in the vascular bundles and cotyledonous parenchyma. As primordia formed, the IAA signal became localized in the root primordia and gradually disappeared in the meristematic zones. In emerging roots, the IAA signal was mainly localized in the root cap and root meristem. These results suggest that accumulation of IAA in the provascular bundles may induce vascular differentiation and the increase in IAA through meristematic zones may be responsible for the adventitious root formation from walnut cotyledons. The direct evidence presented here indicates that IAA accumulated in the meristematic zones is not the sole signal needed to induce adventitious root.