Poly (ADP-ribose) polymerase (PARP) modulates several cellular functional proteins by a mechanism in which the proteins are poly-ADP-ribosylated by transferring the ADPribose moieties from the enzyme substrate NAD+ to the proteins. PARP is activated following damage to cellular DNA by alkylating agents including sulfur mustard (SM). We observed concurrent activation of PARP and DNA ligase in SM-exposed human epidermal keratinocytes (HEK). Previous reports from other laboratories suggested that DNA ligase activation could be due to its modification by PARP. In humans, there are three distinct DNA ligases, I, II and IV of which DNA ligase I participates in DNA replication and repair. By metabolically labeling HEK using 3H-adenosine (adenosine 2,8-3H) to generate intracellularly radiolabeled NAD+ (3H-adenine) and then exposing the cells containing 3H-NAD+ to 1 mM SM, we found that isolated DNA ligase I was not 3Hlabeled. This result indicates that DNA ligase I is not a substrate for ADP-ribosylation by PARP. Interestingly, our results show an effect of PARP inhibition on the decay of activated DNA ligase following exposure of HEK to SM. In the presence of 2 mM 3- amino benzamide (3-AB), a PARP inhibitor, the activated DNA ligase has a half-life that is four-fold higher than that observed in the absence of 3-AB. SM-induced DNA damage repair is dependent on DNA ligase I activation, which we have found to occur via phosphorylation catalyzed by DNA-dependent protein kinase (DNA-PK). The longer half-life of DNA ligase I observed when PARP is inhibited suggests that DNA repair requires PARP, and that DNA ligase I remains activated until DNA damage repair is complete. Therefore, PARP is involved in DNA repair mechanisms other than that of DNA ligase I activation. PARP inhibitors have been suggested as prospective SM antidotes. We are interested in developing mechanism-based intervention of SM injury and, therefore, the knowledge about how PARP participates in DNA repair should be useful for this objective. By using the DNA ligase I phosphorylation assay and inhibiting PARP chemically and also in a separate system through induction of PARP antisense mRNA in the cells, we have confirmed that DNA ligase I is not the target of PARP action. Based on our results and the information available in the literature, we propose that in DNA repair, PARP participates at the stage of altering the chromosomal structure to make the damage accessible to the repair enzymes. Overall, the results of our studies suggest the interplay of PARP and DNA ligase I in the repair of chromosomal DNA damaged by SM.