Apoptotic protease activating factor-1 (Apaf-1) is an established apoptotic protein, but a recent report has indicated that Apaf-1 also plays a role in the DNA damage response. It was shown that Apaf-1 depletion disrupts the ATR-Chk1 pathway and thereby compromises the S-phase checkpoint. This jeopardizes genomic integrity and, indeed, Apaf-1 deficiency has been shown to cause chromosomal instability. The underlying mechanism remains to be resolved. Using UV, camptothecin and laser micro-irradiation as DNA damage inducers we attempt to clarify this matter, showing that depletion of Apaf-1 does inhibit the phosphorylation of Chk1, Nbs1 and RPA32 by ATR, but not ATM, confirming specificity of Apaf-1 to the ATR pathway. Apaf-1 depletion abrogates camptothecin-induced S-phase arrest in U2OS cells, confirming that Apaf-1 deficiency results in a defective S-phase checkpoint. Laser-induced double strand breaks in Apaf-1 depleted U2OS cells in S-phase showed a significant reduction of both RPA and ATR at DNA damage foci compared to control cells. This indicates that ATR is not properly recruited to the site of damage in the absence of Apaf-1. Preliminary data indicate that Apaf-1 is required for the modification of different DNA structures into single stranded stretches, which are necessary for recruitment and activation of ATR.