We have studied the regional distribution of 5-hydroxytryptamine (5-HT) receptor subtypes in fresh circular segments of human cerebral, middle meningeal, and temporal arteries. Vasomotor responses induced by a series of 5-HT agonists and antagonists with some degree of selectivity were studied by using a sensitive in vitro system. Nine 5-HT agonists were examined for contractile effects on the arteries. In cerebral and meningeal arteries 5-carboxamidotryptamine (5-CT) was more potent than 5-HT. The opposite order of potency (5-HT-5-CT) was found in temporal arteries. In the cerebral arteries 5-methoxytryptamine (5-MeOHT) was more potent than sumatriptan while sumatriptan was more potent than 5-MeOHT in meningeal and temporal arteries. The 5-HT1 receptor antagonist, methiothepin, competitively antagonized 5-CT-induced contractions in cerebral arteries, with a pA2 value of 9.05. 5-HT-induced contractions were competitively antagonized by ketanserin (5-HT2) in the temporal arteries pA2 value of 9.06). Methiothepin and ketanserin had non-competitive antagonistic effects in the middle meningeal arteries. The 5-HT3 selective antagonist ondansetron did not cause any shift of the contractions induced by 2-methyl-5-HT in the temporal, cerebral and middle meningeal arteries. These results suggest that the cerebral arteries mainly contain 5-HT1D or 5-HT1-like receptors, and the temporal artery 5-HT2 receptors; the data further indicate the presence of both receptor subtypes in the middle meningeal artery.