Newly developed chemical methods have been applied to study ADP-ribosyl transferase reactions in intact cells following DNA damage. The intracellular levels of NAD and protein-bound monomeric and polymeric ADP-ribose residues were measured in cultured human cells following UV irradiation and in cultured mouse cells treated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). UV irradiation of cells caused a rapid increase in the levels of poly(ADP-ribose). Individual molecules of the polymer are present only transiently and the overall rate of conversion of NAD to poly(ADP-ribose) is proportional to the cellular content of DNA strand breaks. Treatment of cells with MNNG also causes a rapid increase in the levels of both monomers and polymers of ADP-ribose. Non-toxic levels of members of two different classes of ADP-ribosyl transferase inhibitors prevent recovery of cell division following treatment of C3H10T1 /2 cells with MNNG, while closely related compounds that are not inhibitory have no effect. These studies demonstrate that DNA damage results in a rapid perturbation of ADP-ribose metabolism and suggest that ADP-ribosyl transferase activity is necessary for cellular recovery from DNA damage.