Abstract: Inositol pyrophosphates are involved in a variety of cellular functions but the specific pathways and/or downstream targets remain poorly characterized. In this study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by reactive oxygen species (ROS). Yeast lacking kcs1 (the S. cerevisiae IP6K) have greatly reduced IP7 and IP8 levels, and display increased resistance to cell death caused by hydrogen peroxide (H2O2) consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53 controlled functions such actin polymerization appear unaffected by inositol pyrophosphates. Yeast lacking vip1 (S. cerevisiae PP-IP5K) accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7-Kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is likely the double pyrophosphorylated form of IP8 ((PP)2-IP4) that mediates the H2O2 response. However, inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by pleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphates levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of KCS1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionary conserved class of enzymes.