Alkaline-heat-treated titanium self-forms an apatite surface layer in vivo. The aim of the present study was to materialistically characterize the surface of alkaline-heat-treated titanium immersed in simulated body fluid (AHS-TI) and to examine the differentiation behavior of osteoblasts on AHS-TI. SEM, thin-film XRD, FTIR, and XPS analyses revealed that AHS-TI contained a 1.0- micro m-thick, low-crystalline, and [002] direction-oriented carbonate apatite surface. Human osteoblast-like SaOS-2 cells were cultured on polystyrene, titanium, and AHS-TI, and RT-PCR analyses of osteogenic differentiation-related mRNAs were conducted. On AHS-TI, the expression of bone sialoprotein mRNA was up-regulated as compared with that on polystyrene and titanium (p < 0.05). On AHS-TI, the expression of osteopontin and osteocalcin mRNAs was up-regulated as compared with that on polystyrene (p<0.05). The results indicate that the apatite was bone-like and accelerated the osteogenic differentiation of SaOS-2, suggesting that alkaline-heat treatment might facilitate better integration of titanium implants with bone.