Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.