The formation of ATP produced from poly(ADP-ribose) [(ADP-R)_n] has been suggested to be required to repair damaged DNA. Here we investigate whether this ATP is involved in DNA replication processes during DNA repair. Poly(ADP-ribosyl)ated mid-S phase cell nuclei, which were isolated from synchronized HeLa S3 cells followed by the treatment with a DNA damaging agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), were revealed to retain DNA replication synthesizing activity during preincubation for de-poly(ADP-ribosyl)ation only in the presence of pyrophosphate (PPi) before DNA synthesis was started by adding 3 mM ATP. This DNA replication activity was not maintained in the presence of a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase (PARG), Oenothein B (Oen B) during the preincubation with PPi. In the preincubation with PPi, μM orders of ATP was produced from (ADP-R)_n. These results point to an important function of ATP generated from (ADP-R)_n in nuclei for the maintenance of replication apparatus during DNA repair.