Alpha (alpha)-actinin-3 is located in the skeletal muscle Z-line and forms actin-actin crosslinks. An interesting property of alpha-actinin-3 is its expression pattern, which is restricted to fast type II skeletal muscle fibers. However, little is known about the response of alpha-actinin-3 levels to changes in skeletal muscle such as fiber type transformation. This study examined alpha-actinin-3 levels in the soleus muscles of rats subjected to hindlimb unloading, which causes a slow-to-fast fiber transformation in the soleus muscle. After unloading, type II myosin heavy chain (MyHC) and fast myosin levels increased significantly (P<0.0001 for type II MyHC, P<0.005 for fast myosin). Along with these increases in fast fibers, alpha-actinin-3 expression levels increased significantly (P<0.0007) and dramatically. These results indicate that alpha-actinin-3 levels increase concomitantly with increases in skeletal muscle fast fibers.