Neuropeptide Y (NPY) is found to be costored with norepinephrine (NE) in vesicles of the nerve terminals. An endogenous inhibitor of dopamine-beta-hydroxylase (DBH), the synthetic enzyme of NE, has been mentioned. In an attempt to clarify the effect of NPY on DBH activities, an in vitro assay is carried out using chromatographic analysis of NE formation from dopamine. NPY (20-80 pmol/ml) produced a dose-dependent depression of NE formation catalysed by the purified bovine adrenal DBH. Lineweaver-Burke plots (Km = 1.1 mM, Vmax = 10 pmol/min/mg protein) showed a non-competitive inhibition in NPY (30 pmol/ml, IC50)-treated samples. Moreover, failure of denatured NPY even at maximum concentration to influence the DBH activities suggested the essential of natural form of NPY. Participation of sulfhydryl compound seems also negligible, because N-ethylmaleimide did not overcome the effect of NPY. These results indicate that NPY has the ability to inhibit the catalytic action of DBH.