The objective of this study was to examine the effects of myostatin, MyoD and myogenin on apoptosis after hindlimb suspension (HS). A clip, stuck onto the tail with sticky tape, was attached to a suspension apparatus above the cage, allowing full use of the forelimbs and 360 deg free rotation. HS is known to atrophy hindlimb muscles and also induce apoptosis in skeletal muscle of rodent. A variety of transcription factors and cytokines have been demonstrated to have essential roles in regulating apoptosis. One of myogenic regulatory factors, MyoD, and a member of TGF-super family, myostatin, reportedly arrest the cell cycle and upregulate the apoptotic signal transduction in vitro. Moreover, increased expressions of MyoD, myogenin and myostatin are observed during HS. The roles of these proteins have not been examined previously. We raise the hypothesis that MyoD, myogein and myostatin associate with apoptosis during HS. 5-week-old male Wistar rats (n=32) were divided into HS (1 week; n=8, 2 weeks; n=8) and control (1 week;n=8, 2 weeks; n=8) groups. Western blotting of soleus was performed to examine the expressions of Bax and Bcl-2 protein. TUNEL (TdT-mediated dUTP-biotin nick-end labelling) method was conducted first and then each of immunohistochemistry of MSTN, MyoD and myogenin was examined to determine the number of immunoreactive nuclei. Bax and Bcl-2 protein expressions were upregulated in soleus muscle during 7 days of HS. The number of TUNEL positive nuclei relative to muscle cross sectional area increased after HS. Immunoreactive nuclei of myostatin revealed that the ratio of TUNEL positive and myostatin positive nuclei per myostatin positive nuclei in unloaded muscle was higher than that in control. TUNEL-MyoD immunofluorescent staining indicated that the percentage of TUNEL positive and MyoD immunoreactive nuclei out of all MyoD stained nuclei increased after HS. The percent of TUNEL-myogenin immunopositive nuclei in myogenin positive nuclei was found to increase during HS. Bax protein content increased during HS, incicating that HS upregulated the apoptotic signal transduction. These data contributed the increased TUNEL positive nuclei. Myostatin was highly coexpressed with TUNEL staining during unloading. MyoD and myogenin staining increased in TUNEL positive nuclei after HS. During HS myostatin, MyoD and myogenin are possibly recruited to induce apoptosis in rat hinblimb muscle. We should pay attention for the percentages of immunopositives of these two proteins in TUNEL nuclei since the percentages were relatively low. Furthermore, because whole muscle cross sectional area was used to quantify apoptosis, the possibility remains that other cell types contributed to the apoptotic reaction. The findings of this study provide data that help to elucidate molecular mechanisms responsible for atrophy during unloading in rodents. We need to investigate expressions of other factors of apoptosis signals and to test the outcomes from inhibitions of MyoD and myostatin during unloading for further research.