In vitro translation of the RNA isolated from a human pheochromocytoma demonstrated that this tumor contained a mRNA encoding a 10.5-kDa protein, which was immunoprecipitated with antiserum raised against porcine neuropeptide Y. Double-stranded cDNA was synthesized from total RNA and inserted into the Pst I site of pUC8. Transformants containing the neuropeptide Y cDNA were identified using the mixed hybridization probe d[A-(A,G)-(A,G)-T-T-(A,G,T)-A-T-(A,G)-T-A-(A,G)-T-G]. The probe sequences were based on the known amino acid sequence, His-Tyr-Ile-Asn-Leu, found in porcine neuropeptide Y. The nucleotide sequence of the cDNA was determined and contained 86 and 174 bases in the 5'- and 3'-untranslated regions, respectively. The coding sequence consisted of 291 bases, suggesting a precursor to neuropeptide Y that was 97 amino acids long (10,839 Da). The deduced amino acid sequence of the precursor suggested that there were at least two sites of proteolytic processing, which would generate three peptides having 28 (signal peptide), 36 (human neuropeptide Y), and 30 (COOH-terminal peptide) amino acid residues. A partial NH2-terminal sequence obtained by Edman degradation of the immunoprecipitated in vitro translation product identified the positions of methionine and leucine in the first 30 residues of the prepropeptide. A highly sensitive single-stranded complementary mRNA hybridization probe specific for neuropeptide Y mRNA was prepared using the bacteriophage SP6 promoter. This probe was used to identify a mRNA corresponding to neuropeptide Y of approximately 800 bases.