The transforming growth factor-b (TGF-b) network of ligands and intracellular signalling proteins is a subject of intense interest within the field of skeletal muscle biology. To define the relative contribution of endogenous TGF-b proteins to the negative regulation of muscle mass, we used local injection of adeno-associated viral vectors (AAVs) encoding ligand-specific antagonists into the tibialis anterior (TA) muscles of C57Bl/6 mice. Eight weeks after AAV injection, inhibition of activin A and activin B signalling produced moderate (~20%), but significant, increases in TA mass, indicating that endogenous activins repress muscle growth. Inhibiting myostatin induced a more profound increase in muscle mass (~45%) demonstrating a more prominent role for this ligand as a negative regulator of adult muscle mass. Remarkably, co-delivery of activin and myostatin inhibitors induced a synergistic response, resulting in muscle mass increasing by as much as 150%. Transcription and protein analysis indicated that this substantial hypertrophy was associated with both the complete inhibition of the Smad2/3 pathway, and activation of the parallel bone morphogenetic protein (BMP)-Smad1/5 axis (recently identified as a positive regulator of muscle mass). Analyses indicated that hypertrophy was primarily driven by an increase in protein synthesis, but a reduction in ubiquitin-dependent protein degradation pathways was also observed. In models of muscular dystrophy and cancer cachexia, combined inhibition of activins and myostatin, increased mass or prevented muscle wasting, respectively, highlighting the potential therapeutic advantages of specifically targeting multiple Smad2/3-activating ligands in skeletal muscle.