In skeletal muscle of the adult mammal the IIx is a pivotal myosin heavy chain (MHC) isoform that can be either up- or down-regulated depending on both the fiber-type of the target muscle and the type of external stimulus imposed. Since little is known about promoter elements of the IIx MHC gene that are important for its transcriptional regulation in vivo, the main goal of this study was to characterize IIx MHC promoter activity and identify potential regulatory elements. Promoter reporter constructs were transfected into intact rat soleus and plantaris muscle under control and denervated conditions, as well as hindlimb suspension (e.g., models to upregulate IIx MHC transcription). The fast (plantaris) muscle fibers were confirmed to have significantly greater IIx MHC transcriptional products (pre-mRNA and mRNA) than slow (soleus) muscle fibers. However, promoter sequences corresponding to -2671 to +1720, -1000 to +392, -605/+392 relative to the IIx MHC transcription start site, plus an additional contruct ligated to a putative embryonic MHC enhancer, failed to produce a fiber-type specific response that is characteristic of the endogenous IIx MHC promoter. Further, the activity of these promoter constructs did not demonstrate the expected response to denervation or hindlimb-suspension (i.e. marked upregulation), despite normal uptake and activity of a co-injected -actin reference promoter. Based on these findings with IIx MHC promoter reporters we conclude that the loss of the native chromatin environment as well as other necessary cis elements may preclude use of the gene transfer approach, thereby suggesting that there are hidden layers of regulation for the IIx MHC gene.