In this paper, we present the results of systematic studies of the effect of Si substrate preparation on the surface morphology and surface chemistry of indium oxide nanolayers deposited by the rheotaxial growth combined with in situ oxidation under high vacuum under different oxidation regimes controlled using the atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) methods. The AFM experiments showed that Si substrates prepared by standard chemical cleaning procedure, proposed at Radio Corporation of America (RCA) and after ion bombardment cleaning are almost flat characterized by an average roughness below 0.2 nm. For all the deposited indium oxide nanolayers the granular-type surface morphology of different grain sizes was observed. For the most flat one-step oxidized indium oxide (IO) nanolayers deposited on RCA Si substrate there is the highest contribution of elemental In, which is in contrary to the smallest one for the two-step oxidized IO nanolayers deposited at RCA Si substrate, as determined by XPS experiments. (c) 2012 Elsevier B.V. All rights reserved.