We previously showed that chronic cold exposure inhibits eNOS expression and decreases nitric oxide (NO) production. The aim of the present study was to evaluate the possible role of the NO system in the development of cold-induced hypertension (CIH) by testing the hypothesis that adenoviral delivery of human eNOS gene increases NO production and attenuates CIH in rats. The effect of in vivo delivery of adenovirus carrying human eNOS full length cDNA (rAdv.heNOS) on CIH was tested by using 4 groups of Sprague-Dawley rats (6 rats/group). Blood pressure (BP) did not differ among the 4 groups during the control period at room temperature (24°C). Two groups of rats received intravenous injection of rAdv.heNOS (1X10⁹ pfu/rat) and the other 2 groups received the same dose of rAdv.LacZ to serve as controls. After gene delivery, 1 rAdv.heNOS-treated group and 1 rAdv.LacZ-treated group were exposed to cold (6°C), while the remaining groups were kept at 24°C. We found that BP of the rAdv.LacZ group increased significantly within 1 week of exposure to cold, reached a peak level at week 5 (152.2±6.4 mmHg). In contrast, BP (118.7±8.4 mmHg) of the cold-exposed rAdv.heNOS group did not increase until 5 weeks after exposure to cold. rAdv.heNOS increased plasma and urine levels of NO significantly in cold-exposed rats, indicating that eNOS gene transfer increased NO production. Notably, rAdv.heNOS decreased plasma level of norepinephrine and plasma renin activity in cold-exposed rats, suggesting that eNOS gene transfer may decrease the activities of the sympathetic nervous (SNS) system and the renin-angiotensin system (RAS). Immunohistochemical analysis showed that the transferred human eNOS expressed in both the endothelium and adventitia of mesenteric arteries. Conclusions: (1) eNOS gene transfer attenuates CIH by increasing NO production and inhibiting the SNS and the RAS. (2) The NO system appears to mediate this non-genetic, non-pharmacological, non-surgical model of hypertension.