Single-fiber (n = 3,818 fibers) electrophoretic analyses were used to delineate the separate and combined effects of hyperthyroidism (T₃) and hindlimb suspension (HS) on the myosin heavy chain (MHC) isoform composition (1-, 2-, and 4-wk time points) of the rat soleus muscle. The key findings of this study are as follows. First, T₃ and HS both altered the distribution of MHC isoforms at the single-fiber level; however, the populations of fibers produced by these two interventions were clearly different from one another. Second, T₃ + HS rapidly converted the soleus into a fast muscle, producing large increases in the relative contents of the fast type IIx and IIb MHC isoforms which were primarily expressed in several populations of hybrid fibers (e.g., types I/IIa/IIx, I/IIx/IIb, I/IIa/IIx/IIb). Finally, T₃ + HS produced unique populations of hybrid fibers that did not adhere to the I↔IIa↔IIx↔IIb sequential scheme of MHC plasticity. Collectively, the findings of this study demonstrate that the intervention of T₃ + HS is a powerful model for manipulating and studying MHC isoform plasticity in slow skeletal muscle.