We previously found that sustained ERK activation contributes to toxicity elicited by the parkinsonian neurotoxin 6-hydroxydopamine. In addition, substantia nigra neurons from patients with incidental Lewy body disease, Parkinson's disease (PD), and diffuse Lewy body dementia (DLB) display abnormal phospho-ERK accumulations in the form of discrete cytoplasmic granules. In this study, we investigated the subcellular localization of phospho-ERK immunoreactive granules using double label confocal microscopy and immunoelectron microscopy. A small percentage of phospho-ERK granules colocalized with the early endosome marker Rab5, but not with cathepsin D, 20S proteasome β-subunit, or cytochrome P450 reductase. Phospho-ERK immunoreactivity was often associated with mitochondrial proteins (MnSOD, 60 kDa and 110 kDa mitochondrial antigens), and some vesicular-appearing phospho-ERK granules appeared to envelop enlarged mitochondria by confocal laser scanning microscopy. Ultrastructural immuno-gold studies revealed phospho-ERK labeling in mitochondria and in association with bundles of ∼10 nm fibrils. Heavily labeled mitochondria were observed within autophagosomes. As mitochondrial pathology may play a pivotal role in Parkinson's and other related neurodegenerative diseases, these studies suggest a potential interaction between dysfunctional mitochondria, autophagy, and ERK signaling pathways.