The midbrain periaqueductal grey matter (PAG) has numerous functional roles that include mediating nociceptive inhibition and integrating behavioural and physiological responses to potentially threatening or stressful stimuli. Underlying these behaviours is the diverse interconnectivity of this region, and it is possible that neurochemical subdivisions within the PAG reflect the functional properties of the different PAG regions. In this study, using in situ hybridization, we have investigated the distribution in the rat PAG of the messenger ribonucleic acids (mRNAs) encoding seven neuropeptides: enkephalin (ENK), substance P (SP), somatostatin (SST), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Each peptide mRNA had a distinct topographical distribution in the PAG. Preproenkephalin A (ENK) mRNA-expressing cells were found at all levels of the PAG in three distinct longitudinal columns. Preprotachykinin A (SP)-expressing cells were found at all levels of the PAG, principally in the Edinger-Westphal nucleus and the lateral and dorsal PAG. There was a column of neurones producing mRNA-encoding somatostatin that extended along the rostrocaudal extent of the ventrolateral PAG; there were also labelled cells in the dorsal and dorsolateral subdivisions at some levels of the PAG. Galanin mRNA-producing neurones were limited to the dorsal raphe nucleus and to a second population in the ventral border of the aqueduct. VIP mRNA-producing neurones were found in very localized regions of the PAG, including the cell-sparse region immediately ventral to the aqueduct and the ventral part of the dorsal raphe nucleus. NPY mRNA-producing neurones were localized mainly in some cells of the Edinger-Westphal nucleus and dorsal raphe nucleus. CGRP mRNA-expressing neurones were limited to the oculomotor and trochlear nucleus. The results showed a topographical distribution of neuropeptides over the rostrocaudal extent of the PAG that is compatible with the emerging theory that the anatomical and functional specificity of the PAG is expressed in the form of longitudinally arranged neuronal columns that extend for varying distances along the rostrocaudal axis of the midbrain PAG. © 1994 Wiley-Liss, Inc.