The arbuscules of mycorrhizae develop within apoplastic compartments of the host plant, as they are separated from the cell protoplast by an interfacial matrix continuous with the plant cell wall. Expansins are proteins that allow cell wall loosening and extension. Using fluorescence and electron microscopy we located the NtEXPA5 epitopes recognized by polyclonal antibody anti-NtEXPA5 in mycorrhizal tobacco roots. The expansin protein was localized mainly within the interfacial matrix of intracellular hyphae, arbuscule trunk and main branches. NtEXPA5 proteins were detected neither within the interface of collapsing arbuscule branches nor in non-colonized cortex cells. In plant cell walls, expansin protein was detected only at the penetration point and in the parts of cell walls that adhered firmly to fungal hyphae growing intracellularly. For the first time, NtEXPA5 protein was localized ultrastructurally in hyphae growing intracellularly at the interface of the hypha tip and sites of bending. The novel localization of NtEXPA5 protein suggests that this protein may be involved in the process of arbuscule formation: that is, in promoting apical hyphal growth and arbuscule ramification, as well as in controlling the dynamic of arbuscule mycorrhiza development.