Stimulation of osteoblast proliferation and differentiation is important for the in vivo bone-bonding ability of biomaterials. Previous in vitro studies have used biochemical assays to analyze osteoblast-specific gene expression in cultured osteoblasts. In this study, we generated transgenic mice harboring a monomeric red fluorescent protein 1 transgene under the control of a 2.3-kb fragment of the Col1a1 promoter, which is active specifically in osteoblasts and osteocytes. We established a fluorescent primary osteoblast culture system to allow noninvasive observation of osteoblast proliferation and differentiation on opaque materials in vitro. We used this system to evaluate alkali- and heat-treated titanium, which has a strong bone-bonding ability in vivo, and we observed a rapid increase in fluorescence intensity and characteristic multifocal nodule formation. A cell proliferation assay and RT-PCR to examine osteoblast-specific gene expression showed increased osteoblast proliferation and differentiation consistent with the fluorescence observations. This mouse model allowed us to use fluorescence intensity to visualize and quantify in vivo newly formed bone around implanted materials in femurs. The use of these fluorescent osteoblasts is a promising method for simple screening of the bone-bonding ability of new materials.