Abstract Pharmacological evidence has suggested the presence of 5-hydroxytryptamine (5-HT, serotonin), 5-HT1D receptors on endothelial cells but these receptors have never been identified unambiguously on this type of cells. We now report that human umbilical vein endothelial cells (HUVEC) express 5-HT1D receptors coupled to inhibition of cyclic AMP formation. 5-HT and the 5-HT1D receptor agonists 5-carboxamidotryptamine (5-CT) and sumatriptan were approximately equipotent at inhibiting forskolin-stimulated cyclic AMP accumulation in HUVEC (mean pEC50 7.68.2, maximal effect 30% inhibition). The 5-HT1A receptor agonist, 8-OH-DPAT was clearly less potent (pEC50 6.2) and less efficacious. The selective 5-HT1D receptor antagonist, GR127935 (1nM) markedly inhibited the effect of 5-HT (apparent pKB 10.8). Reverse transcription-polymerase chain reaction analysis showed the mRNA for 5-HT1D receptors to be expressed in HUVEC. These results demonstrate the presence of functional 5-HT1D receptors and the expression of 5-HT1D receptor mRNA in HUVEC. They support the involvement of 5-HT1D receptors in endothelial-mediated responses to 5-HT.