Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including alpha5 integrin and vinculin, was higher in 3D compared with 2D (p,0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p,0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of alpha-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5+/-0.8 to 7.4+/-4.7 kPa during proliferation (p,0.05) and reached 12.2+/-6.0 kPa during differentiation (p,0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.