The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the idealtissue-engineering scaffold (i.e. histocompatibility, porosity, degradability, non-toxicity). Wepreviously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment invivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenicenvironment, regardless of its location, we analysed MAS interaction with both muscle and nonmusclecells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogeneticprotein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro,thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supportedformation of nascent muscle fibres when replacing a muscle (orthotopic position). However,heterotopically grafted MAS did not give rise to muscle fibres when transplanted within the renalcapsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoidprocess, in spite of the abundant presence of cells migrating along the laminin-based MASstructure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenicdifferentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to theintrinsic properties of the muscle scaffold (e.g. specific muscle ECM proteins). Indeed, it is morelikely that myogenic stem cells colonising MAS recognise a muscle environment that ultimatelyallows terminal myogenic differentiation. In conclusion, MAS may represent a suitableenvironment for muscle and non-muscle 3D constructs characterised by a highly organised structurewhose relative stability promotes integration with the surrounding tissues. Our work highlights theplasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissueengineering applications than the mere replacement of volumetric muscle loss.