Neuronal nitric oxide synthase (nNOS) catalyzes production of nitric oxide in vertebrate brains. Recent findings indicate that endothelial NOS and reactive oxygen species (ROS) are significantly increased during hypoxic stress and are modulated by antioxidants. However, the influence of antioxidants and steroids on nNOS upregulation by hypoxia is largely unknown. In this study, we characterized nNOS cDNA and examined the effects of hypoxia and antioxidant and steroid treatments on nNOS expression in Atlantic croaker hypothalamus. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 and/or 4weeks) caused significant increases in hypothalamic nNOS mRNA, protein and its neuronal expression. Hypothalamic nNOS expression and superoxide radical (O2(·-), an index of ROS) production were increased by pharmacological treatment of fish exposed to normoxic conditions with N-ethylmaleimide, an alkene drug which covalently modifies sulfhydryl groups and inhibits aromatase activity. In contrast, treatments with Nω-nitro-L-arginine methyl ester, a competitive NOS-inhibitor, or vitamin E, an antioxidant, prevented the upregulation of O2(·-) production and nNOS expression in hypoxia-exposed (DO: 1.7 mg/L for 4 weeks) fish. Moreover, treatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, increased hypothalamic O2(·-) production and nNOS expression in normoxic control fish; whereas estradiol-17β treatment significantly reduced O2(·-) production and nNOS expression in hypoxia-exposed fish. Double-labeled immunohistochemical results showed that nNOS and aromatase proteins are co-expressed in the hypothalamus. Taken together, the results suggest that upregulation of nNOS and ROS in the croaker hypothalamus in response to hypoxia is influenced by antioxidant and overall estrogen status.