Quantitative real-time PCR and Griess reaction were conducted to evaluate gene expression of nitric oxide synthases (eNOS, nNOS and iNOS) and nitric oxide (NO) production in the hindbrain, midbrain, forebrain of chickens with cold-induced pulmonary hypertension. The ratio of the right to total ventricular weight of heart as an indication of pulmonary hypertension was increased in the cold stress groups of chickens at 42 d of age. In the pulmonary hypertensive chickens, production of NO was increased in the hindbrain but was unchanged in the forebrain and midbrain. Relative gene expression of eNOS and nNOS was upregulated in the three segments of brain, whereas the iNOS transcript was downregulated in the forebrain and midbrain of the cold-induced pulmonary hypertensive chickens. It is concluded that in the chickens with cold-induced pulmonary hypertension, variations of nNOS, eNOS and iNOS gene expression would lead to overproduction of NO in the hindbrain, whereas the variations in the expression of these genes did not result in an elevation of NO in the forebrain and midbrain. It is suggested that high levels of NO in the hindbrain excites neural mechanisms involved in pulmonary hypertension.