Several focal skeletal muscle diseases, including tumours and trauma lead to a limited loss of functional muscle tissue. There is still no suitable clinical approach for treating such defects. A promising approach could be the tissue engineering of skeletal muscle. However, a clinically reliable differentiation stimulus for three-dimensional (3-D) cultures is necessary for this process, and this condition has not yet been established. In order to qunantify and analyze the differentiation potential of electrical cell stimulation, primary myoblasts were stimulated within a 3-D fibrin-matrix. Gene expression of MyoD, myogenin and AChR were measured by real-time RT-PCR over a time period of eight days, showing immediate down-regulation of all marker genes. For tissue engineering approaches, cell multiplication is crucial for acquisition of sufficient tissue volumes for reconstruction. Therefore, all experiments were performed with high and low passaged myoblasts, demonstrating higher transcript rates of marker genes in lowpassage cells. Our findings strongly suggest a reconsideration of electrical stimulation in muscle tissue engineering.