Skeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha–smooth-muscle actin (α-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.