The peels of citrus fruits are a rich source of flavonoids, which have been shown to exert beneficial properties on human health. Nobiletin (5,6,7,8,3′,4′- hexamethoxyflavone) is a major component of polymethoxylated flavones found in the peel of citrus fruits, and is used in a Chinese traditional herbal medicine. Nobiletin exhibits a broad spectrum of pharmacological activities, including anticancer, antioxidative, and anti-inflammatory properties. Furthermore, nobiletin possesses antiatherogenic and cardiovascular protective effects, and neuronal effects such as neurotrophic effects in vitro, and anti-dementia activities in vivo, although the precise mechanisms underlying these nobiletin’s effects remain to be determined. Adrenal medullary cells derived from the embryonic neural crest are functionally homologous to the sympathetic postganglionic cells. In cultured bovine adrenal medullary cells, the Na+ influx induced by acetylcholine (ACh) via nicotinic ACh receptor-ion channels is a prerequisite for Ca2+ influx via the activation of voltage-dependent Ca2+ channels and subsequent catecholamine synthesis and secretion. Stimulation of catecholamine synthesis induced by ACh is associated with an activation of tyrosine hydroxylase, which catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA), the rate-limiting step of catecholamine biosynthesis. We previously demonstrated that nobiletin enhances catecholamine secretion and Ca2+ influx in cultured bovine adrenal medullary cells (J Neurochem., 114: 1030-1038, 2010). Here we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of 14C-catecholamines from [14C]tyrosine in a time (20-30 min) and concentration (1.0 -100 microM)-dependent manner. Nobiletin (10-100 microM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on 14C-catecholamine synthesis was not observed when extracellular Ca2+ was deprived from the incubation medium. Several protein kinase inhibitors such as H-89 and KN-93, inhibitors of cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II, respectively, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. On the other hand, nobiletin (1.0-100 microM) inhibited 14C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis and tyrosine hydroxylase activity, whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.