In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.