Rhabdomyosarcomas (RMS) are the most common soft tissue cancer among children and are characterized by their expression of the myogenic regulatory factors MyoD and myogenin. Yet RMS cells cannot undergo normal myogenesis and are caught between the proliferation program and the terminal differentiation program. Many questions still remain about the defects present in rhabdomyosarcoma cells. In this work, we set out to understand the role of myogenin in these cells. To begin, we found that myogenin and its co-factors were present in rhabdomyosarcoma cells at levels that should support terminal differentiation. We examined the expression profile of several myogenin target genes in rhabdomyosarcoma cells and then assayed for myogenin activity using luciferase reporter constructs that contain myogenin dependent promoters to test for myogenin function. Many myogenin target genes were down regulated in RMS cells but that the target promoters on the luciferase constructs were activated. Terminal differentiation is a complicated process that involves many proteins. In cancer cells, it is important to compare the levels proteins with known functions to those levels in wild-type cells at the protein and RNA levels. Establishing the defect of rhabdomyosarcoma cells can lead to further insights into normal myogenesis, and may also lead to new therapeutic approaches in the treatment of this childhood cancer.