This study characterizes the relaxant response to 5-hydroxytryptamine (5-HT) in prostaglandin F(2alpha) (PGF(2alpha))-precontracted pulmonary arteries of weaned pigs. In arterial rings with intact endothelium, the relaxation to 5-HT was biphasic. The high affinity component of relaxation to 5-HT (0.1-10 nM) was abolished by mechanical removal of the endothelium or after the addition of L: -NAME (200 microM), and was inhibited by the 5-HT(2B/2C) receptor antagonist SB 206553 (1 microM), but not the 5-HT(2C) receptor antagonist SB 242084 (0.1 microM). Endothelium-intact arteries were also relaxed by the selective 5-HT(2B) receptor agonist BW 723C86 (pD(2) 7.7). The relaxant response to BW 723C86 was inhibited by 1 microM SB 206553 (pK(B) 6.8). The low affinity component of relaxation to 5-HT (>/=30 nM) remained unaffected after mechanical removal of the endothelium or the addition of L: -NAME. In endothelium-denuded arterial rings, 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), and frovatriptan produced monophasic relaxations with pD(2) values of 6.5, 7.5, 5.9, and 4.7 respectively. Relaxant responses to the agonists were antagonized by the selective 5-HT(7) receptor antagonist SB 269970 (pK(B) 8.2-8.9). The relaxant response to the potent 5-HT(7) receptor agonist 5-CT was also antagonized by methiothepin (pK(B) 9.6), pimozide (pK(B) 8.2), mesulergine (pK(B) 7.7), methysergide (pK(B) 7.4), clozapine (pK(B) 7.6), and spiperone (pK(B) 7.4). The estimated pK(B) values argue in favor of an involvement of 5-HT(7) receptors in the direct vasorelaxant action of 5-HT in the pulmonary arteries of weaned pigs. The relaxant response to 5-CT was associated with an increase in cAMP that was surmountably antagonized by SB 269970 (pK(B) 8.6). The present in vitro bioassay can be used to characterize new drugs with potential agonist or antagonist properties at functional 5-HT(7) receptors.