The c-myc gene encodes a sequence-specific DNA binding protein involved in proliferation and oncogenesis. Activation of c-myc expression in quiescent cells is sufficient to mediate cell cycle entry, whereas inhibition of c-myc expression causes cycling cells to withdraw from the cell cycle. To search for components of the cell cycle machinery that are targets of c-Myc, we have made a mutant c-Myc protein, named MadMyc, that actively represses c-myc target genes. Expression of MadMyc in cycling NIH3T3 cells causes a significant accumulation of cells in G1. The MadMyc-induced G1 arrest is rescued by ectopic expression of cyclin E/CDK2 and cyclin D1/ CDK4, but not by Cdc25A, a known cell cycle target of c-Myc. The MadMyc G1 arrest does not require the presence of a functional retinoblastoma protein and is associated with a strong reduction in cyclin E/CDK2 kinase activity in arrested cells. MadMyc does not cause alterations in the expression levels of cyclin E, CDK2, p27kip1, cyclin D1 or CDK4 in G1-arrested cells. These data indicate that inhibition of c-Myc activity in exponentially growing cells leads to G1 arrest through loss of cyclin E-associated kinase activity.