Abstract Neuropeptide Y (NPY) is found to be costored with norepinephrine (NE) in vesicles of the nerve terminals. Tyrosine hydroxylase (TH), the synthetic enzyme of NE, has been mentioned to be a rate-limiting step. In an attempt to understand the effect of NPY on TH activities, an in vitro assay is carried out using chromatographic analysis of 3,4-dihydroxyphenylalanine (DOPA) formation from tyrosine. NPY (40 120 pmol/ml) produced a dose-dependent depression of DOPA formation catalysed by the adrenal TH of rats. Lineweaver-Burk plot ( K m = 156 μM. V max = 1.05 nmol/h/mg protein) showed a non-competitive inhibition in NPY (80 pmol/ml, IC 50)-treated samples. Moreover, failure of denatured NPY even at maximum concentration to influence the TH activities suggested the essential of nature form for NPY. Participation of pterine cofactor seems also negligible, because increase of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine did not overcome the effect of NPY. These results indicate that NPY has the ability to inhibit the catalytic action of TH in the adrenal gland of rats.