Ultrasound frequency of 1.0 MHz was applied with an ultrasound probe to a piece of excised hairless mouse skin for enhancing the rate of prednisolone (PN) transfer across the skin in vitro. The results showed that the rate of PN transfer through the intact skin was greatly enhanced according to the application time of ultrasound. Although ultrasound increases the temperature adjacent to the probe, its effect on enhancing the rate of PN transfer through the intact skin was found to be insignificant. Pretreatment experiments in which ultrasound was applied before performing normal permeation experiments were also performed. Although ultrasound was not applied in the normal permeation experiments following the pretreatment, the results showed that pretreatment also enhanced the rate of PN transfer through the intact skin in proportion to the period of pretreatment time. This implied that the ultrasound might have changed some structures of the skin. Pretreatment of 1800 s or more made the rate of PN transfer through the intact skin close to that through tape-stripped skin. This was caused either by a morphology change of the skin or by enlargement of the diameters of some follicles, or by both.