The interactions of neuropeptide Y with dimyristoylphosphatidylcholine and cell membranes were examined by several physical techniques to probe the potential role of its putative C-terminal amphipathic alpha-helix. Neuropeptide Y binding was demonstrated by a rapid release of entrapped 6-carboxyfluorescein and a rapid decrease in the turbidity of dimyristoylphosphatidylcholine liposomes. In addition, an increase in tyrosine fluorescence intensity and an increase in the anisotropy of diphenylhexatriene in dimyristoylphosphatidylcholine liposomes was observed. In isolated, aortic smooth muscle cell membranes, the anisotropy of diphenylhexatriene increased as a function of added neuropeptide Y. The concentration range (low microM) over which neuropeptide Y increases the polarization of diphenylhexatriene in cell membranes is similar to the range in which it inhibits isoproterenol-stimulated cAMP accumulation. This inhibition is not affected by pertussis toxin, nor does neuropeptide Y cause the release of preloaded [3H]adenine from cells into the medium. These data suggest that neuropeptide Y contains an amphipathic alpha-helical region which interacts with lipids in much the same way as the amphipathic alpha-helical regions of the plasma apolipoproteins and that the inhibition of isoproterenol-stimulated cAMP accumulation at low microM concentrations of peptide may be the result of an alteration in the cell membrane bilayer structure.