Researchers have attempted to enhance titanium osseointegration by modifying its surface properties, including via H(2)O(2) pre-treatment, with reported treatment regimes varying from minutes/hours, to weeks. This study examined the effects of various H(2)O(2) treatments on titanium surface topography/roughness, chemical composition/oxide thickness, hydrophilicity and plasma protein adsorption. Titanium discs were treated with 30% H(2)O(2) for 0-24 h or 1-4 weeks and subjected to atomic force microscopy (AFM), scanning electron microscopy (SEM), profilometry, X-ray photon spectroscopy and contact angle analysis. For protein adsorption, whole plasma and FITC-conjugated serum albumin were added to 0-24 h and 1-4 week H(2)O(2)-treated discs and examined by SEM and fluorescence microscopy, respectively. AFM, SEM and profilometry demonstrated that 1-6 h H(2)O(2)-treated discs exhibited subtle alterations in surface topography/roughness at the nanometre scale, although 24 h and 1-4 week H(2)O(2)-treated discs exhibited much greater increases in surface roughness, in the micrometre range. Maximal increases in surface oxide thickness and chemical modification were identified between 1 h-4 weeks and 3 h-4 weeks, respectively, although no increases in oxygen/titanium (O1s : Ti2p) molar ratio or in hydrophilicity were evident. Plasma and serum albumin adsorption increased on 1-24 h H(2)O(2)-treated discs, with further increases on 1-4 week H(2)O(2)-treated discs. Based upon the present data and previous findings, this study supports the concept that surface topography/roughness and oxide composition/thickness, are more significantly modified by H(2)O(2) treatment and more influential to protein adsorption than hydrophilicity. Additionally, it can be hypothesized that the 24 h H(2)O(2) treatment of titanium surfaces, which induced micrometre scale changes in roughness and protein adsorption, to those associated with enhanced osteoblast attachment/behaviour, mineralisation and subsequent implant osseointegration, would be most beneficial. To cite this article:Nagassa ME, Daw AE, Rowe WG, Carley A, Thomas DW, Moseley R. Optimisation of the hydrogen peroxide pre-treatment of titanium: surface characterisation and protein adsorption.Clin. Oral Impl. Res. 19, 2008; 1317-1326doi: 10.1111/j.1600-0501.2008.01611.x.