Basic helix-loop-helix (bHLH) proteins mediate terminal differentiation in many lineages. By using the bHLH protein MyoD, which can dominantly activate the myogenic differentiation program in numerous cell types, we demonstrated that recessive defects in bHLH protein function are present in human tumor lines. In contrast to prior work with primary cell cultures, MyoD did not activate the myogenic program in six of the eight tumor lines we tested. Cell fusions between the MyoD-defective lines and fibroblasts restored MyoD activity, indicating that the deficiency of a gene or factor prevents bHLH protein function in the tumor lines. Fusions between certain pairings of the MyoD-defective lines also restored MyoD activity, allowing the tumor lines to be assigned to complementation groups on the basis of their ability to execute the myogenic program and indicating that multiple mechanisms exist for abrogation of bHLH protein activity. These groups provide a basis for identifying genes critical for bHLH-mediated differentiation and tumor progression by using genetic complementation.