This study was designed to investigate the optimal period of pedicles implantation in the prefabricated periosteofascial flap with a vascular tissue transfer. The flap prefabrication was prepared with a transposition of left occipital pedicles on the calvarial fascia of male Sprague-Dawley rats. Thirty flaps were divided into five groups of six flaps, including control group (group I) of the conventional periosteofascial flap based on the lateral border of the rat calvarium. The prefabricated flap was elevated as an $1{ imes}1cm$ sized island flap based on the implanted pedicle at 1, 2, 3, and 4 weeks after the pedicles transfer in groups II, III, IV, and V, respectively. After the completion of creating a critical-sized calvarial defect and implanting with hydroxyapatite granules, the flap was sutured back for covering the defect and kept isolated from surrounding tissues. Six weeks after flap repositioning, the osseous changes of the defect were examined with simple radiographic findings, radiodensitometric analysis, and histological studies. By simple radiographic findings, specimens of the control, groups IV and V showed homogeneous radioopacity within the defect. But in groups II and III, focal radiolucency was observed in the defect. In the radiodensitometric analysis, the control group and the group V showed significant increased radiodensites statistically. Histologically, the implanted hydroxyapatite was absorbed partly in the defect in groups II, III, and IV. In the defects of the control group and the group V, the implanted hydroxyapatite was kept in its volume and the deposition of the bone cells was observed sparsely. In conclusion, the prefabricated periosteofascial flap can be created with a vascular tissue transfer and the pedicles should be implanted at least for 4 weeks to bring out positive osseous changes in the calvarial defect.