We report on the function of the human ortholog of Saccharomyces cerevisiae Rif1 
(Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates 
their length. In contrast, human Rif1 did not accumulate at functional 
telomeres, but localized to dysfunctional telomeres and to telomeric DNA 
clusters in ALT cells, a pattern of telomere association typical of 
DNA-damage-response factors. After induction of double-strand breaks (DSBs), 
Rif1 formed foci that colocalized with other DNA-damage-response factors. This 
response was strictly dependent on ATM (ataxia telangiectasia mutated) and 
53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related 
kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition resulted in 
radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase 
checkpoint phenotype was independent of Nbs1 status, arguing that Rif1 and Nbs1 
act in different pathways to inhibit DNA replication after DNA damage. These 
data reveal that human Rif1 contributes to the ATM-mediated protection against 
DNA damage and point to a remarkable difference in the primary function of this 
protein in yeast and mammals.