Muscle disuse induces substantial alterations in the highly plastic skeletal muscle tissues, which occur especially in antigravity slow muscles. We differentially screened a muscle cDNA array to identify modifications in gene profile expression induced in slow rat soleus muscle mechanically unloaded by hindlimb suspension as a model for muscle disuse. This study focused on muscle creatine kinase mRNA and protein and glyceraldehyde-3-phosphate dehydrogenase mRNA, which were found to be upregulated in unweighted muscles. These upregulations were analyzed over a 4-wk time course of hindlimb suspension and compared with variations in myosin heavy chain (MHC) isoforms while specifically focusing on type IIx MHC mRNA and protein. The two metabolic marker upregulations clearly preceded IIx MHC contractile protein upregulation. Muscle creatine kinase upregulation was shown to be an excellent, and the earliest, marker of muscle disuse at mRNA and protein levels.