Although large-scale approaches have identified numerous mitochondrial phosphoproteins, little is known about the mitochondrial kinases and phosphatases that regulate these phosphoproteins. Here, we identify two members of the atypical dual specificity phosphatases (DSP), DSP18 and DSP21, that are localized in mitochondria. Although DSP18 is widely expressed in several mammalian tissues, DSP21 is selectively expressed in the testes. We demonstrate that DSP18 and DSP21 are targeted to mitochondria by cryptic internal localization signals. Subfractionation of mitochondria demonstrated that DSP18 is located in the intermembrane space as a peripheral membrane protein of the inner membrane. In contrast, subfractionation of rat testis mitochondria revealed DSP21 is localized to the matrix as a peripheral membrane protein of the inner membrane. Moreover, we demonstrate that a previously reported substrate for DSP18, the stress-activated protein kinase, does not localize to mitochondria in several different tissues, making it an unlikely substrate for DSP18. Finally, we show that induction of apoptosis by treatment with staurosporine causes translocation of DSP18 from the intermembrane space into the cytosol similar to other apoptogenic factors such as cytochrome c. This work rigorously demonstrates the unique location of two highly similar DSPs on opposing sides of the mitochondrial inner membrane.