Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type I myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the down-regulation of MHC I in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC I promoter was injected into soleus muscles of control rats and those subjected to seven days of hindlimb suspension (HS). Mutation analyses of six putative regulatory elements within the -408 bp region demonstrated that three elements: an A/T-rich, the proximal MCAT (-e3), and an E-box (-63bp) play an important role in the basal level of MHC I gene activity in the control soleus and function as unloading-responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the -e3 and E-box probes with nuclear extract from HS soleus compared to control soleus. Supershift assays indicated that TEF-1 and myogenin factors bind the -e3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the TEF-1 and myogenin factors were significantly attenuated in the unloaded soleus compared to the control muscle. We conclude that the down-regulation of MHC I in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements.