Titanium dioxide (TiO2) is widely used as a white pigment in paints, plastics, inks, paper, creams, cosmetics, drugs and foods. In the present study, the genotoxicity of anatase TiO2 nanoparticles was evaluated in vivo using the comet assay after a single or repeated intratracheal instillation in rats. The nanoparticles were instilled intratracheally at a dosage of 1.0 or 5.0mg/kg body weight (single instillation group) and 0.2 or 1.0mg/kg body weight once a week for 5weeks (repeated instillation group) into male Sprague–Dawley rats. A positive control, ethyl methanesulfonate (EMS) at 500mg/kg, was administered orally 3h prior to dissection. Histopathologically, macrophages and neutrophils were detected in the alveolus of the lung in the 1.0 and 5.0mg/kg TiO2 groups. In the comet assay, there was no increase in % tail DNA in any of the TiO2 groups. In the EMS group, there was a significant increase in % tail DNA compared with the negative control group. TiO2 nanoparticles in the anatase crystal phase are not genotoxic following intratracheal instillation in rats.