To clarify how muscle length or tension influences protein balance, we examined rates of protein synthesis and degradation in rat muscles incubated at different lengths. In soleus and extensor digitorum longus, protein degradation was lowest at resting length in situ (Lo), where the muscles are under slight tension. Muscles that were unrestrained shortened spontaneously and showed 25-45% greater net protein breakdown than muscles fixed at Lo. Protein synthesis did not differ in the two groups initially, but by 4 or 6 h was 25% greater in muscles at Lo. Maintenance of Lo improved overall protein balance largely through the reduction in proteolysis. In addition, muscles at Lo maintained high levels of ATP and phosphocreatine for 6 h, whereas the unrestrained muscles lost about half their energy stores. The acceleration of proteolysis in the shortened muscle appears independent of prostaglandin E2. However, the acceleration of protein breakdown in the unrestrained muscles could be prevented with inhibitors of thiol proteases (e.g., Ep-475 or leupeptin) or by incubation in Ca2+-free medium or with dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum). These treatments did not reduce the low level of proteolysis in muscles at Lo. Thus, if muscles are allowed to shorten in vitro, proteolysis is activated by a Ca2+-dependent mechanism, involving thiol proteases.