AbstractWe have recently reported that thermal oxidation treatments of Ti6Al4V at 500° and 700°C for 1 h result in the formation of an outer “ceramic” layer of rutile that do not decrease the high in vitro corrosion resistance of the alloy. In the present work, surface roughness was measured and found marginally increased as a consequence of oxidation of the alloy at 700°C, but not at 500°C. We have evaluated the biocompatibility of the oxidized surfaces, by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared with polished alloy, both thermal treatments increased osteoblast adhesion measured as cell attachment, β1 integrin and FAK‐Y397 expression, as well as cytoskeletal reorganization. Compared with treatment at 500°C, thermal oxidation at 700°C enhanced cell adhesion. Treatment at 700°C transiently impaired cell proliferation and viability, which were not altered in alloys oxidized at 500°C. Several markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, and mineralized nodule formation were found either unaffected or differentially increased by alloys treated either at 500° or 700°C. In addition, thermal oxidation at 700°C also increased osteoprotegerin secretion. Taken together, our results indicate that thermal oxidation treatments at 500° or 700°C for 1 h improve the in vitro biocompatibility of Ti6Al4V. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 73A: 97–107, 2005