Cellular prion protein (PrP) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrP misfolds to a pathogenic isoform PrP, the causative agent of neurodegenerative prion diseases. The precise function of PrP remains elusive but may depend upon its cellular localization. Here we show that PrP is present in brain mitochondria from 6-12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrP was fully processed with mature N-linked glycans and did not require the GPI anchor for localization. Protease treatment of purified mitochondria suggested that mitochondrial PrP exists as a transmembrane isoform with the C-terminus facing the mitochondrial matrix and the N-terminus facing the intermembrane space. Taken together, our data suggest that PrP can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrP may affect mitochondrial function.