A standard animal model for phototoxicity evaluation does not appear in any guideline. Sprague-Dawley (SD) rats have been widely used in general toxicity and toxicokinetic studies and can be used in phototoxicity evaluation to reduce animal usage. To standardize phototoxicity procedures of SD rat, we investigated skin site- and sex-related differences in sensitivity to drug-induced phototoxicity at 3 facilities. Six-week-old male and female SD rats were orally administered 30 or 100 mg/kg lomefloxacin and light irradiation 1 hr after dosing; an ultraviolet (UV) irradiation device (10 J/cm2, UVA) or solar simulator (18 J/cm2, UVA) was used as light sources. Phototoxic reactions on ventral skin, dorsal skin, and auricle were observed macroscopically at 2, 24, 48, and 72 hr after irradiation. Plasma concentrations of lomefloxacin were also measured in non-irradiated, conscious rats. Skin reaction scores for ventral skin were highest and those of dorsal skin were lowest among the skin sites examined at all dose levels and facilities. Although drug concentrations in plasma were almost similar between sexes or higher in males than females, skin reaction scores appeared higher in females than males for ventral or dorsal skin. A difference in skin reaction scores among facilities was also observed; however, the order of skin sites based on sensitivity was approximately the same. We therefore suggest that appropriate conditions be drafted at each facility as differences in sensitivity to phototoxicity are dependent on skin site or sex in SD rats. Furthermore, we encourage multi-site validation studies to standardize experimental conditions in in-vivo phototoxicity studies.