1. The effect of functional overload produced by tenotomy of synergistic gastrocnemius muscle on the expression of myosin heavy chain (MHC) isoforms in the plantaris and soleus muscles of the rat was studied using gradient sodium dodecyl sulfate-acrylamide gel electrophoresis. 2. Five weeks tenotomy, the plantaris and soleus muscle weights induced by tenotomy of the gastrocnemius muscle were 44.3%(P< 0.005) and 37.4%(P< 0.005), respectively, heavier than the contralateral control muscles. 3. Although four types of MHC isoforms were observed in both control and experimental plantaris, the percentage of MHC isoforms in the control and experimental muscles differed; the hypertrophied plantaris muscle contained more HCI (P< 0.05), HCIIa and HCIId (P< 0.05) and less HCIIb (P< 0.05) than the control muscle. 4. The control soleus muscle contained two MHC isoforms, HCI and HCIIa. However, there was only a single HCI isoform in the hypertrophied soleus muscle. 5. These results indicate that overloading a skeletal muscle by removing its synergists produces not only the muscle hypertrophy but also the changes in the expression of MHC isoforms.