Moderate hypothermia (25–31 °C) may have a significant influence on vascular tone. At present, very little is known about the role of endothelial nitric oxide on the hypothermia-induced responses. In this study, we investigated the effect of hypothermia (to 28 °C) on the vasodilatation induced by verapamil, a phenylalkylamine calcium channel blocker (10−9–3 × 10−4 M) and dihydropyridines, amlodipine (10−9–3 × 10−4 M), and benidipine (10−9–10−3 M) on 5-hydroxytryptamine (5-HT or serotonin) precontracted calf cardiac veins. Furthermore, the role of nitric oxide in the hypothermia-induced responses was analyzed. Ring preparations of veins obtained from calf hearts were suspended in organ baths containing 15 ml of Krebs–Henseleit solution, maintained at 37 °C, and continuously gassed with 95% O2–5% CO2. After a resting period, verapamil, amlodipine, and benidipine were applied cumulatively on serotonin (10−6 M) precontracted calf cardiac vein rings and induced concentration-dependent relaxations. In another part of the study, the medium temperature was decreased to 28 °C after the preparations were contracted with 5-HT, then cumulative concentrations of verapamil, amlodipine, or benidipine were added. During hypothermia, the pIC50 value, but not the maximal response, to all blockers were significantly higher than at 37 °C. Hypothermia in the presence of NG-nitro-l-arginine methyl ester (L-NAME, 10−4 M) decreased the pIC50 and Emax values to verapamil, amlodipine, and benidipine. Only one blocker was tested in each preparation. These results suggest that nitric oxide may play a role in the hypothermia-induced changes in vasodilation caused by verapamil, amlodipine, and benidipine in calf cardiac vein, but further research is needed to explain the complete mechanism.