Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (P_o), but decrease the specific P_o (sP_o) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of EDL and soleus muscles from wild type (MSTN^+/+), heterozygous-null (MSTN^+/-) and homozygous-null (MSTN^-/-) adult male mice were determined. For EDL muscles, the P_o of both MSTN^+/- and MSTN^-/- mice were greater than the P_o of MSTN^+/+ mice. For soleus muscles, the P_o of MSTN^-/- mice was greater than that of MSTN^+/+ mice. The sP_o of EDL muscles of MSTN^-/- mice was less than MSTN^+/+ mice. For soleus muscles, however, no difference in sP_o was observed. Following two lengthening contractions, EDL muscles from MSTN^-/- mice had a greater force deficit than MSTN^+/+ or MSTN^+/- mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, ActRIIB. Compared with the soleus, the amount of ActRIIB was approximately two-fold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles, but also in the contractility and the composition of the ECM of muscles.