Abstract: Using a combination of TAP (tandem affinity purification) tagging and mass spectrometry, we have characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin sensitive complex) which plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C, a known regulatory subunit PP4R2, and a novel protein that we have termed PP4R3. The S. cerevisiae PP4R3 ortholog Psy2 was previously identified in a screen for sensitivity to the DNA damaging agent and anticancer drug cisplatin. We demonstrate here that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicits cisplatin hypersensitivity. Further, human PP4R3 complements the yeast psy2 deletion, and D. melanogaster lacking functional PP4R3 (flfl) exhibit cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally, we find that PP4R3 may target PP4cs to the DNA-damage repair machinery at least in part via an interaction with Rad53 (CHK2).