Existing models describing sarcomere assembly have arisen primarily from studies using cardiac muscle. In contrast to cardiac muscle, skeletal muscle differentiation is characterised by dramatic changes in protein expression, from non-muscle to muscle-specific isoforms before organisation of the sarcomeres. Consequently, little is understood of the potential influence of non-muscle cytoskeletal proteins on skeletal sarcomere assembly. To address this issue, transfectant (γ33-B1) and control mouse C2 myoblasts were differentiated to form myotubes, and various stages of skeletal sarcomere assembly were studied. Organisation of non-muscle γ-actin and co-localisation with sarcomeric α-actinin, an early marker of sarcomere assembly and a major component of Z lines, was noted. γ-Actin was also identified in young myotubes with developing sarcomeric myofibrils in regenerating adult mouse muscle. Localisation of γ-actin in a different area of the myotube to the muscle-specific sarcomeric α-actin also indicated a distinct role for γ-actin. The effects of aberrant γ-actin expression in other myoblast lines, further suggested a sequestering role for γ-actin. These observations make the novel suggestion that non-muscle γ-actin plays a role in skeletal sarcomere assembly both in vitro and in vivo. Consequently, a modified model is proposed which describes the role of γ-actin in skeletal sarcomere assembly.