Ribonucleotide reductase (RR) is a rate-limiting enzyme that catalyzes de novo conversion of ribonucleotide 5'-diphosphates to the corresponding 2'-deoxynucleotide, essential for DNA synthesis and replication. The mutations or knockout of RR small subunit, p53R2, results in the depletion of mitochondria! DNA (mtDNA) in human, implying that p53R2 might play a critical role for maintaining mitochondria] homeostasis. In this study, siRNA against p53R2 knockdown approach is utilized to examine the impact of p53R2 depletion on mitochondria and to derive underlying mechanism in KB and PC-3 cancer cells. Our results reveal that the p53R2 expression not only positively correlates with mtDNA content, but also partakes in the proper mitochondria function, such as ATP synthesis, cytochrome c oxidase activity and membrane potential maintenance. Furthermore, overexpression of p53R2 reduces intracellular ROS and protects the mitochondrial membrane potential against oxidative stress. Unexpectedly, knockdown of p53R2 has a modest, if any, effect on mitochondrial and total cellular dNTP pools. Taken together, our study provides functional evidence that mitochondria is one of p53R2-targeted organelles and suggests an unexpected function of p53R2, which is beyond known RR function on dNTP synthesis, in mitochondrial homeostatic control. (C) 2011 Elsevier Inc. All rights reserved.