Myostatin (GDF-8) is the chief chalone in skeletal muscle and negatively controls adult skeletal muscle growth. The role of myostatin during overload-induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would result in reduced myostatin protein levels. Overload-induced hypertrophy was accomplished by unilateral tenotomy of the gastrocnemius tendon in male adult Sprague-Dawley rats followed by a two-week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active, latent and precursor myostatin protein levels. Significant hypertrophy was noted in the plantaris (494 ± 29 vs. 405 ± 15 mg, p < 0.05) and soleus (289 ± 12 vs. 179 ± 37 mg, p < 0.05) muscles following overload. Overloaded soleus muscle decreased the concentration of active myostatin protein by 32.7 ± 9.4% (p < 0.01) while the myostatin precursor protein was unchanged. Overloaded plantaris muscle decreased the concentration of active myostatin protein by 28.5 ± 8.5% (p < 0.01) while myostatin precursor levels were reduced by 17.5 ± 5.9% (p < 0.05). Myostatin latent complex concentration decreased in the overloaded soleus and plantaris muscle by 15.0 ± 5.9% and 70.0 ± 2.3% (p < 0.05), respectively. These data support the hypothesis that the myostatin signaling pathway in overloaded muscles is generally downregulated and contributes to muscle hypertrophy. Plasma concentrations of total and active myostatin proteins were similar in overloaded and control animals and averaged 8865 ± 526 pg/ml and 569 ± 28 pg/ml, respectively. Tissue levels of BMP-1, an extracellular proteinase that converts myostatin to its active form, also decreased in overloaded soleus and plantaris muscles by 40.4 ± 12.9% and 32.9 ± 6.9% (p < 0.01), respectively. These data support the hypothesis that local, rather than systemic, regulation of myostatin contributes to the growth of individual muscles, and that an association exists between the extracellular matrix proteinase BMP-1 and the amount of active myostatin in overloaded muscles.