Neuropeptide Y (NPY) is the only member of its peptide family that has been isolated from the mammalian CNS. We have recently found that two different NPY-related molecules are present in the CNS of a cyclostome, the river lamprey (Lampetra fluviatilis) (Soderberg et al., 1991). Here we show that this is also true for the rat CNS, by demonstrating expression of peptide YY (PYY) mRNA in brainstem neurons distinct from those neurons that express NPY mRNA. Dissimilar oligonucleotide DNA probes complementary to 3' untranslated regions of the rat PYY, NPY, and pancreatic polypeptide (PP) mRNA were used in in situ hybridization experiments on sections of rat brain and spinal cord, visceral organs, and peripheral nerve ganglia. The PYY probe hybridized with two populations of neurons in the brainstem: one dispersed along the midline in the rostral medulla and another in the lateral caudal medulla (A1 region). No additional labeling was detected in the remainder of the neuraxis. In the periphery, PYY hybridization was seen only in endocrine cells of the colon, and not in sympathetic ganglia or the adrenal gland, suggesting that previous observations of PYY immunoreactivity in these latter structures were due to antibody cross- reactivity with NPY. The NPY probe did not hybridize with cells on the midline region that contains PYY neurons, but it did label large numbers of neurons throughout the neuraxis. No expression of PP mRNA was detected in the CNS. Northern blot analysis failed to detect PYY mRNA in the CNS, further supporting the observation that PYY is only expressed by a discrete collection of CNS neurons. The anatomy of PYY- and NPY-expressing cells in the CNS and gut shows a striking similarity between rat and lamprey (Brodin et al., 1989), vertebrates that diverged evolutionarily about 450 million years ago, suggesting that both peptide systems have been conserved throughout vertebrate evolution.