Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15 INK4b by binding to the initiator element of the p15 INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15 INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15 INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15 INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.