For a cell to proliferate in cancer, and during normal development or tissue maintenance, it must replicate its chromosomal DNA. We are investigating the regulation of DNA replication in human cells and cell extracts at the molecular level. Using these approaches, we have characterised the small non-coding Y RNAs as essential factors for the initiation step of human chromosomal DNA replication in vitro. Y RNAs are also required for DNA replication and proliferation of intact cells in culture, and they are significantly over-expressed in solid human tumours. The molecular mechanisms by which Y RNAs regulate the initiation of chromosomal DNA replication are still unknown. Addressing these mechanisms, we have recently identified an essential conserved motif for Y RNA function, as well as molecular interactions between Y RNAs, the pre-replication complex and unreplicated DNA. Our data obtained so far suggest that Y RNAs act as licensing factors for the control of DNA replication.