Despite the important contribution of the midbrain periaqueductal gray (PAG) to endogenous pain suppression systems, little is known about the neuroanatomical basis of its functional organization. In a previous study of the distribution of the endogenous opiate leucine-enkephalin (ENK) in the PAG (Moss, M. S., E. J. Glazer, and A. I. Basbaum (1983) J. Neurosci. 3: 603–616), we found that immunoreactive ENK-containing neurons and terminals are clustered in discrete populations. In this study we have extended our analysis of the neurochemical organization of the PAG by using immunocytochemistry to map the distribution of two non-opiate peptides that produce potent analgesia when administered at central gray levels: substance P (Sub P) and vasoactive intestinal polypeptide (VIP). Immunoreactive Sub P neurons and terminal fields are clustered in discrete populations throughout the PAG. The distribution pattern of these populations changes at different rostral-caudal levels of the PAG. For example, there is a ventral-to-dorsal shift in the location of Sub P-like immunoreactivity from the caudal to the rostral PAG. Few immunoreactive Sub P neurons are found in the nucleus raphe dorsalis although moderately dense terminal field staining is present. The staining pattern of immunoreactive VIP is totally different from that of Sub P. Regardless of the rostral-caudal level examined, VIP- containing neurons are found tightly clustered in the subependymal neuropil of the ventromedial PAG. Only a few immunoreactive VIP- containing neurons are found in the ventral PAG or nucleus raphe dorsalis. The striking differences between the distribution of Sub P- and VIP-like immunoreactivity in the PAG indicates that the neural circuitry underlying pain suppression by Sub P and VIP may also differ.