Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes proline-containing peptides shorter than 30-mer. It has been suggested that POP is associated with cognitive functions and inositol 1,4,5-triphosphate (IP3) signaling. However, little is known about the distribution and physiological role of POP in the brain. We used immunohistochemistry to determine the cellular and subcellular distribution of POP in the rat brain. POP was specifically expressed in the glutamatergic pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and also in the CA1 field of hippocampus. Purkinje cells of the cerebellum were also intensively immunostained for POP. Double immunofluorescence indicated that POP was present in the γ-aminobutyric acid (GABA)ergic and cholinergic interneurons of the thalamus and cortex but not in the nigrostriatal dopaminergic neurons. POP did not colocalize with astrocytic markers in any part of the rat brain. We used postembedding immunoelectron microscopy to determine the distribution of POP at the subcellular level. POP was mainly present in neuronal cytosol and membranes, hardly at all in neuronal plasma membrane, but more extensively in intracellular membranes such as the rough endoplasmic reticulum and Golgi apparatus. Our findings point to a role for POP—evidently modifying neuropeptide levels—in excitatory and inhibitory neurotransmission in the central nervous system via glutamatergic, GABAergic, and cholinergic neurotransmission systems. Furthermore, according to our results, POP may be involved in thalamocortical neurotransmission, memory and learning functions of the hippocampal formation, and GABAergic regulation of voluntary movements. Subcellular distribution of POP points to a role in protein processing and secretion. J. Comp. Neurol. 507:1694–1708, 2008. © 2008 Wiley-Liss, Inc.