In this work we investigated the physical and chemical nature of ultraflat titanium dioxide (TiO2) samples which we had previously used as substrates for the investigation of adsorbed protein molecules (Cacciafesta, P.; Humphris, A. D. L.; Jandt, K. D.; Miles, M. J. Langmuir 2000, 16, 8167). Titanium films were prepared by thermal evaporation on a heated mica surface and either separated from the mica to investigate the resulting surface or left in contact with the mica to analyze the titanium−mica interface. Atomic force microscopy (AFM) of the surfaces exposed after removal of the mica showed very flat surfaces [root-mean-square roughness of 0.29 nm ± 0.03 nm] and the presence of disordered as well as ordered structures. Different degrees of order were observed, such as a square lattice with spacing of a 0 = 0.49 nm, a regular pattern with spacings of a 0 = 0.47 nm and b 0 = 0.35 nm with an angle of ∼80° between a 0 and b 0, or the presence of surface defects. These ordered structures were not stable upon long-term AFM imaging and could be damaged by the scanning tip. To further investigate the nature of the ordered regions, X-ray diffraction (XRD) was performed for the titanium−mica samples. XRD showed the crystalline structures of both the mica and the titanium film but did not detect any order in the titanium−mica interface. Possible causes for the formation of the ordered regions are discussed, in the cases of formation both after removal of mica or during sample preparation. The chemical nature of the titanium−mica interface was investigated with secondary ion mass spectrometry and X-ray photoelectron spectroscopy (XPS) via depth profiling of the titanium−mica samples from the titanium side. The metal film was found to be mainly composed of titanium and to a lesser extent of oxygen and carbon. After the metal film was sputtered and the titanium−mica interface was reached, the titanium concentration decreased and the concentration of the typical mica elements, such as silicon, aluminum, and potassium, increased. XPS was also used to investigate the chemical composition of the surfaces obtained after mica removal, which were found to be mainly composed of TiO2 with a small percentage of Ti2O3. The possible applications of this simple method for preparing ultraflat TiO2 surfaces are discussed.