Cellular prion protein (PrP C) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrP C misfolds to a pathogenic isoform PrP Sc, the causative agent of neurodegenerative prion diseases. The precise function of PrP C remains elusive but may depend upon its cellular localization. Here we show that PrP C is present in brain mitochondria from 6–12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrP C 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 C 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 C can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrP C may affect mitochondrial function.