The purpose of this study was to investigate whether there are any differences in tooth movement or in the response of periodontal tissue to orthodontic force when the force is applied at different times of the day. One hundred 6-week-old male Wistar rats were divided into one control group without force application and three experimental groups based on the time of day the force was applied to the upper first molars. Animals in the whole-day group received force continuously throughout the experimental period, while animals in the light- and dark-period groups received force only during the light (07:00-19:00) or dark period (19:00-07:00), respectively. Tooth movement was measured using the occlusal view of a precise plaster model with a profile projector. Periodontal tissues were evaluated histologically. The time course of tooth movement varied among the groups. Tooth movement over 21 days in the whole-day and light-period groups was about twice that as in the dark-period group. The formation of new bone on the tension side in the whole-day and light-period groups was more than twice that as in the dark-period group. On the pressure side, more osteoclasts appeared on the alveolar bone in the whole-day and light-period groups than in the dark-period group. The light-period group showed less extensive hyalinization of the periodontal ligament (PDL) than the whole-day group. The area of root resorption on day 21 also varied among the groups. Interference by masticatory forces did not seem to be a principal cause of the decreased tooth movement in the dark-period group. These results indicate that there are considerable variations in tooth movement and in the response of periodontal tissue to orthodontic force when the force is applied at different times of the day in rats. The results suggest that diurnal rhythms in bone metabolism have important implications in orthodontic treatment.