The interaction between cooling and vasoactive substances, e.g. 5-hydroxytryptamine (5-HT), plays an important role in the pathophysiology of cold-induced vasospasm. Our objective was to study the effect of cooling on the 5-HT vascular response, classify the involved 5-HT receptors, and to analyze the role of the endothelium. Ring segments from the rat jugular vein, a preparation without α-adrenergic receptors, were suspended in organ baths to record the circular motor activity. The temperature was initially 37 °C and was thereafter either continuously lowered to 10 °C or kept constant at different temperatures within this range. 5-HT at low concentrations (10<sup>–11</sup> to 3 × 10<sup>–8</sup> M) induced relaxation at 37 °C in segments precontracted by prostaglandin F<sub>2α</sub><sup>–</sup> The relaxation was recognized to be mediated via an endothelium-dependent 5-HT<sub>1</sub>-like receptor mechanism presumably involving the release of endothelium-derived relaxing factor (EDRF). Cooling to 29 and 20 °C diminished the relaxation, probably due to an attenuated release of EDRF. 5-HT at concentrations of more than 10<sup>–8</sup> M induced a contraction in all vessels at 37 °C mediated via a 5-HT<sub>2 </sub>receptor. An increased 5-HT-induced contraction was seen at temperatures below 37 °C in vessels with an intact endothelium. Endothelial denudation diminished the cold-induced enhancement of the contraction to 5-HT. These studies suggest that endothelial mechanisms contribute to a cold-induced augmented response to 5-HT.