Glutaredoxin (Grx) is a specific and efficient catalyst of glutathione-dependent deglutathionylation of protein-SS-glutathione mixed disulfides. Grx has been identified in brain cytosol, but the presence of activity in subcellular organelles has not been reported. Increases in protein glutathionylation are likely to occur in mitochondria during oxidative stress and it is, therefore, important to know if this organelle contains the enzyme activity needed to reverse such protein thiolation. Grx-like activity in the P1 supernatant from rat brain and liver was doubled in the presence of Triton-X 100 suggesting a releasable pool of Grx. Brain and liver homogenates were subfractionated into cytosolic, mitochondrial and microsomal fraction, their purity determined by biochemical assay and EM and assayed for Grx-like activity. The data presented demonstrate that mitochondria contain functional Grx-like activity.