Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but there have been few studies conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using the primary culture of chicken embryonic myoblasts, in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1), targeting GDF-8 mRNA was introduced into the myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52 kD GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)₁₀-constructed luciferase reporter assay, a concomitant change in the activity was initiated after 24 h. The activity rapidly decreased 30 h after the siRNA-1 introduction, whereas the high activity was maintained at 30-42 h in the control and siRNA-2. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and their myotubes with the aggregated nuclei were shorter and wider. These results strongly suggest that the deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes.