Various diseases are characterized by oxidative stress, when overproduction of reactive oxygen- and nitrogen-centered intermediates (ROI, RNI) occurs. Reactive species such as peroxynitrite and hydrogen peroxide are capable of causing DNA breakage triggering the activation of the DNA nick sensor enzyme poly(ADP-ribose) polymerase (PARP-1). Activated PARP-1 cleaves NAD+ into nicotinamide and (ADP-ribose), then attaches ADP-ribose residues onto nuclear acceptor proteins, and synthesizes branching polymer chains. PARP-1 fulfills a great number of biological functions ranging from the regulation of DNA repair, chromatin structure, replication, transcription to facilitating telomere elongation. However, overactivation of PARP by ROI and RNI in inflammation, shock and reperfusion injury, leads to necrotic cell death mainly due to depletion of NAD+ and ATP. We have developed three applications: enzyme cytochemistry, enzyme histochemistry and cellular ELISA to detect the activation of poly(ADP-ribose) polymerase in oxidatively stressed cells. and tissues The assays are based on the use of biotinylated NAD+ as PARP substrate. Furthermore, we have investigated the role of poly(ADP-ribose) glycohydrolase (PARG), the main poly(ADP-ribose) catabolyzing enzyme in oxidative stress-induced cytotoxicity. We have found that inhibition of PARG by gallotannin (GT) provided significant cytoprotection to peroxynitrite/hydrogen peroxide treated HaCaT cells (decrease in LDH release, propidium iodide uptake and caspase-3 activation), inhibited depletion of cellular NAD+ pools and caused the accumulation of poly(ADP-ribose) and inhibition of cellular PARP activity. Moreover, we have investigated the possible role of intracellular Ca2+ mobilization and high cell density, in the regulation of sensitivity of HaCaT keratinocytes to oxidative stress-induced cytotoxicity. First we have demonstrated that peroxynitrite triggered an elevation in intracellular calcium levels. Treatment of cells with the cell permeable calcium chelator BAPTA-AM provided significant cytoprotection against peroxynitrite/hydrogen peroxide–induced cytotoxicity. We have also observed that differentiating confluent keratinocytes were highly resistant to oxidative stress. Our results support a crucial role of Ca2+ homeostasis, cell density and poly(ADP-ribose) metabolism in the regulation of oxidative stress-induced cytotoxicity.