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Titanium and its alloys are employed as artificial joints, bone plates, wires, screws and bone prostheses in orthopedic and dental fields, because of their high corrosion resistance, good mechanical properties, and biocompatibility. Since they cannot directly bond to living bone–tissue through stable chemical interactions, a few surface modification techniques have been proposed for giving materials apatite-forming ability that secures bone–tissue bonding, such as chemical treatment with H 2 O 2 or NaOH, electrochemical oxidation, electrophoretic apatite particle deposition, and UV-irradiation of surface titanium oxide layer. This study examined how the combination of H 2 O 2 chemical treatment and UV-irradiation affected in vitro apatite-formation on TiO 2 (anatase phase) layers as UV was irradiated under a few different conditions. TiO 2 layer was prepared by the chemical treatment with H 2 O 2 solution and subsequent heat-treatment (CHT). CHT samples were irradiated with UV-light for 1 h in air or in ultra-pure water. They were then soaked in Kokubo's simulated body fluid (SBF; pH 7.4) at 36.5 °C for 1 day. Their surface structure and morphology were examined by using a thin film X-ray diffractometer (TF-XRD), and a scanning electron microscope (SEM). The UV-irradiation of CHT in air reduced the number of active sites for apatite nucleation. On the contrary, however, the UV-irradiation in water increased them. These opposite results indicate that environmental factors of the UV-irradiation are important for controlling the in vitro apatite-forming ability of anatase layer.