Neuroendocrine secretory protein 55 (NESP55) is a recently characterized secretory protein localized to large dense-core vesicles resembling the class of chromogranins. We investigated the distribution of the messenger RNA encoding for NESP55 in the rat brain by in situ hybridization with specific ³⁵S-labelled oligonucleotides. NESP55 messenger RNA was detected only on neuronal but not glial cells. In the brain, expression of NESP55 messenger RNA was most prominent in several areas throughout the midbrain and brainstem, including the locus coeruleus, the raphe complex and the reticular formation. NESP55 messenger RNA-expressing cells were also found in many areas and nuclei throughout the hypothalamus. Neocortical areas, the hippocampus and the cerebellum were devoid of NESP55 messenger RNA-containing neurons. From this distribution pattern, a significant overlap of NESP55 expression with the noradrenergic, adrenergic and serotonergic transmitter systems was evident. The present study defines, for the first time, the cellular localizaton of NESP55 messenger RNA in the rat brain. The present results provide the basis for future studies defining the as yet obscure function of NESP55.