The INK4/ARF locus encodes three tumour suppressors (p15(INK4b), ARF and p16(INK4a)) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RD(INK4/ARF)). Targeted and localized RNA-interference-induced heterochromatinization of RD(INK4/ARF) results in transcriptional repression of the locus, revealing that RD(INK4/ARF) is a relevant transcriptional regulatory element. Cdc6 is overexpressed in human cancers, where it might have roles in addition to DNA replication. We have found that high levels of Cdc6 result in RD(INK4/ARF)-dependent transcriptional repression, recruitment of histone deacetylases and heterochromatinization of the INK4/ARF locus, and a concomitant decrease in the expression of the three tumour suppressors encoded by this locus. This mechanism is reminiscent of the silencing of the mating-type HM loci in yeast by replication factors. Consistent with its ability to repress the INK4/ARF locus, Cdc6 has cellular immortalization activity and neoplastic transformation capacity in cooperation with oncogenic Ras. Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16(INK4a). We conclude that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RD(INK4/ARF) element.