Myostatin is a member of the transforming growth factor-β (TGF-β) family that acts as a negative regulator of skeletal muscle mass. This study investigates the regulation of myostatin signaling and identifies potential inhibitors that may promote muscle growth. Myostatin is purified from mammalian cells as a noncovalent complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal dimer binds to activin type II receptors, Act RIIB and Act RIIA. Binding of myostatin to Act RIIB can be inhibited by follistatin and the myostatin propeptide. Transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB showed dramatic increases in muscle mass, similar to those seen in myostatin knockout mice. These findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful for enhancing muscle growth in humans and livestock. Myostatin is expressed in the myotome compartment of developing somites and continues to be expressed in the myogenic lineage throughout development and in adult animals. Mice with a targeted deletion of the myostatin gene have a dramatic increase in skeletal muscle mass. The myostatin sequence is highly conserved across species, and mutations in the myostatin gene result in the double muscling phenotype in cattle. These findings suggest that pharmacological agents capable of blocking myostatin activity may have applications for promoting muscle growth in human disease settings and livestock. The study also explores the regulation of myostatin signaling. Myostatin is normally present in a latent complex with its propeptide and other proteins. Upon activation, myostatin signals by binding to activin type II receptors. The study shows that myostatin can bind to Act RIIB and that this binding can be inhibited by follistatin and the myostatin propeptide. Transgenic mice expressing the myostatin propeptide or follistatin showed increased muscle mass, indicating that these molecules may be useful for enhancing muscle growth. The study also investigates the effect of the myostatin propeptide and follistatin on myostatin activity. The propeptide and follistatin were capable of blocking the binding of the C-terminal dimer to Act RIIB. The K_i of follistatin was estimated to be approximately 470 pM, while that of the propeptide was at least 50-fold higher. These findings suggest that the propeptide and follistatin may be useful for enhancing muscle growth. The study also shows that the most dramatic effects on skeletal muscle were obtained by using the follistatin construct. Two founder animals showed increased muscling, with muscle weights increased by 194–327% relative to control animals. These results suggest that follistatin mayMyostatin is a member of the transforming growth factor-β (TGF-β) family that acts as a negative regulator of skeletal muscle mass. This study investigates the regulation of myostatin signaling and identifies potential inhibitors that may promote muscle growth. Myostatin is purified from mammalian cells as a noncovalent complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal dimer binds to activin type II receptors, Act RIIB and Act RIIA. Binding of myostatin to Act RIIB can be inhibited by follistatin and the myostatin propeptide. Transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB showed dramatic increases in muscle mass, similar to those seen in myostatin knockout mice. These findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful for enhancing muscle growth in humans and livestock. Myostatin is expressed in the myotome compartment of developing somites and continues to be expressed in the myogenic lineage throughout development and in adult animals. Mice with a targeted deletion of the myostatin gene have a dramatic increase in skeletal muscle mass. The myostatin sequence is highly conserved across species, and mutations in the myostatin gene result in the double muscling phenotype in cattle. These findings suggest that pharmacological agents capable of blocking myostatin activity may have applications for promoting muscle growth in human disease settings and livestock. The study also explores the regulation of myostatin signaling. Myostatin is normally present in a latent complex with its propeptide and other proteins. Upon activation, myostatin signals by binding to activin type II receptors. The study shows that myostatin can bind to Act RIIB and that this binding can be inhibited by follistatin and the myostatin propeptide. Transgenic mice expressing the myostatin propeptide or follistatin showed increased muscle mass, indicating that these molecules may be useful for enhancing muscle growth. The study also investigates the effect of the myostatin propeptide and follistatin on myostatin activity. The propeptide and follistatin were capable of blocking the binding of the C-terminal dimer to Act RIIB. The K_i of follistatin was estimated to be approximately 470 pM, while that of the propeptide was at least 50-fold higher. These findings suggest that the propeptide and follistatin may be useful for enhancing muscle growth. The study also shows that the most dramatic effects on skeletal muscle were obtained by using the follistatin construct. Two founder animals showed increased muscling, with muscle weights increased by 194–327% relative to control animals. These results suggest that follistatin may