Both chemical and topographic cues are crucial for the development of skeletal muscle. In this study, the relative roles of both signals in regard to cell adhesion, morphology, and differentiation of C2C12 skeletal myoblasts were investigated. Grooved polystyrene substrates containing grooves with approximately 900?nm in width with 600?nm ridge spans and 665?nm in depth were conjugated with the cell adhesion peptide arginineglycineaspartic acid (RGD). RGD conjugation significantly enhanced the adhesion, growth and differentiation of C2C12 cells. On the other hand, anisotropic topography primarily directed the direction and alignment of myoblasts and myotubes. The results in this study provide information regarding the relative roles of chemical and topographic cues in musculoskeletal myogenesis, and are of interest to applications in muscle tissue engineering. Biotechnol. Bioeng. 2012; 109:21042115. (c) 2012 Wiley Periodicals, Inc.