Double-layered architecture is capable of growing high-aspect-ratio TiO2 nanotubes of small diameter to increase dye loading ability for dye-sensitized solar cells. The tube growth of the second layer is significantly affected by initial seeding sites. However, the seeding mechanism of the bottom nanotubes is still ambiguous. In this work, the seeding sites of the bottom nanotubes are tailored to be at upper tube bottoms, intertube gaps or random position on an interlayer by varying interim treatments. The results reveal that new tubes form preferentially at the location where the oxide layer is etched faster to a critical thickness for pore nucleation. In the presence of an interlayer (e.g., >10 nm), random seeding is induced. A model is thus created to interpret the relevant seeding behaviors. The mechanistic study is useful in tailoring the configuration of double-layered structure for growing high-aspect-ratio nanotubes. (C) 2013 Elsevier Ltd. All rights reserved.