The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34-substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34-substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor.