The eukaryotic genome is replicated according to a specific spatio-temporal 
programme. However, little is known about both its molecular control and 
biological significance. Here, we identify mouse Rif1 as a key player in the 
regulation of DNA replication timing. We show that Rif1 deficiency in primary 
cells results in an unprecedented global alteration of the temporal order of 
replication. This effect takes place already in the first S-phase after Rif1 
deletion and is neither accompanied by alterations in the transcriptional 
landscape nor by major changes in the biochemical identity of constitutive 
heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S 
transition and chromatin re-organization after DNA replication. Together, these 
data offer a novel insight into the global regulation and biological 
significance of the replication-timing programme in mammalian cells.