Cis-acting sequences involved in the expression of the human neuropeptide Y (NPY) gene were identified by transient expression and in vitro transcription assays using the Lan-5 cell system. Sequences between -63 and -51 were necessary for NPY gene expression. Gel retardation analyses utilizing this region indicate that a sequence-specific DNA-binding protein interacts with the sequence CCCCTCC. An in vitro nuclear transcription system was prepared from Lan-5 cell nuclei in order to determine a functional correlation between the CCCCTCC-binding protein and promoter activity. The NPY promoter was accurately transcribed in this system; furthermore, the data obtained from the in vitro transcription experiments were similar to those obtained from the in vivo transfection studies. Competition experiments in which an oligonucleotide containing either CCCCTCC or Sp1 (GGCGGG) sequences was added in excess to the transcription extracts demonstrated the functional importance of an Sp1-like protein in NPY gene transcription.