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Abstract: This study investigated the surface characteristics and in vitro biocompatibility of a titanium (Ti) oxide layer incorporating calcium ions (Ca) obtained by hydrothermal treatment with or without post heat-treatment in the Ti–13Nb–13Zr alloy. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, atomic force microscopy and contact angle measurements. In vitro biocompatibility of the Ca-containing surfaces was assessed in comparison with untreated surfaces using a pre-osteoblast cell line. Hydrothermal treatment produced a crystalline CaTiO3 layer. Post heat-treatment at 400°C for 2h in air significantly decreased water contact angles in the CaTiO3 layer (p <0.001). The Ca-incorporated alloy surfaces displayed markedly increased cell viability and ALP activity compared with untreated surfaces (p <0.001), and also an upregulated expression of various integrin genes (α1, α2, α5, αv, β1 and β3) at an early incubation time-point. Post heat-treatment further increased attachment and ALP activity in cells grown on Ca-incorporated Ti–13Nb–13Zr alloy surfaces. The results indicate that the Ca-incorporated oxide layer produced by hydrothermal treatment and a simple post heat-treatment may be effective in improving bone healing in Ti–13Nb–13Zr alloy implants by enhancing the viability and differentiation of osteoblastic cells. [Copyright &y& Elsevier]