Mitochondrial diseases (MDs) are a group of heterogeneous disorders due to impaired oxidative phosphorylation causing defective ATP production. The histopathological hallmark is the presence ofragged-red fibers (RRFs), muscle fibers with excessive mitochondrial proliferation. Nitric oxide synthases (NOSs) are enzymes responsible of the synthesis of nitric oxide (NO), a ubiquitous signaling molecule involved in many physio-pathological processes. Three NOS isoenzymes have been identified so far including neuronal NOS (NOS1), inducible NOS (NOS2) and endothelial NOS (NOS3). Despite the expression and the subcellular localization of NOS1 and NOS3 have been previously investigated, a possible involvement of NOS2 in MDs has never been assessed. We evaluated the expression of NOS2 in muscle biopsies from 17 patients with mitochondrial respiratory chain dysfunction. Our data demonstrate that NOS2 is overexpressed in RRFs and the correspondence between NOS2 immunoreactivity and SDH staining suggests that the protein localizes to the mitochondria. Together with previous studies from the literature, these findings indicate a possible role NOSs in the pathogenic events leading to MDs