The carboxy-terminal binding protein (CtBP)-interactingprotein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its rolein this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5'flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP thatis deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks. © 2014 Elsevier Inc.