Alpha-synuclein (alpha-Syn) is a brain-enriched protein of 140 amino acids. Despite of strong evidence showing the implication of the protein in the pathogenesis of several neurodegenerative diseases, its physiological function remains poorly understood. To study the physiological function of alpha-Syn, a depiction of its precise subcellular localization is necessary. Although alpha-Syn expression in the brain has been extensively investigated using several different antibodies, its precise subcellular localization in neurons remains elusive. In this study, immunogold electron microscopy with a newly produced 3D5 monoclonal antibody recognizing the C-terminal 115-121 amino acids of alpha-Syn was used to examine its subcellular localization in rat brain neurons. In addition, the relative amount of the protein in different subcellular pools of the neurons in several brain regions was evaluated and compared. The results showed that alpha-Syn-positive gold particles were unevenly distributed in axons, presynaptic terminals, cytoplasm and nucleus in the neuron, with the density of gold particles being greater in presynaptic terminals and nucleus than in other subcellular pools. In the cytoplasmic region, relatively dense gold particles were seen in some mitochondria. In the same subcellular pools, the density of gold particles was varied among the neurons from different brain regions. Although the cortical neurons showed much higher density of gold particles in the presynaptic terminals and nuclei than in striatal, hippocampal and substantia nigral neurons, the density of gold particles in their mitochondria was much lower compared with the mitochondria of striatal, hippocampal and substantia nigral neurons. The relative high level of mitochondrial alpha-Syn in hippocampus, striatum and substantia nigral neurons may have special pathophysiological significance, which deserves further investigation.