The stable culture of myogenic cells and their differentiation into myotubes in vitro is often hindered by the mechanical destabilisation of the spontaneously contractile neotissue formed, resulting in the complete loss of differentiating myotubes. Electrospun, parallel aligned nylon 6/6 microfibre arrays were use successfully for the culture of C2C12 myoblasts and their differentiation to form mechanically stable, orientated myotubes in vitro. Myoblasts adhered strongly to the parallel fibre array, forming a compact cell sheath across the entire array, aligning individual cells in parallel to the direction of the fibrous substratum. The myogenic potential of C2C12 myoblasts was not impaired and resulted in the formation of elongated myotubes expressing alpha-actinin, adult myosin heavy chain and nicotinic acetylcholine receptors as muscle-specific marker proteins. Newly formed C2C12 myotubes were themselves orientated in parallel to the direction of the underlying fibrous substratum and exhibited a high level of structural integration with the surrounding cells. In contrast, non-woven, non-orientated nylon 6/6 meshes, produced by conventional electrospinning, exhibited greatly reduced levels of C2C12 myoblast attachment and adherent myoblasts did not differentiate into myotubes. In conclusion, parallel microfibre arrays provided a superior microscale topography for the stable maintenance and differentiation of myotubes in vitro.