Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mad/Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stimulated the activity of MyoD by approximately 10-fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis.