&NA;It has been shown previously that serotonin stimulates the production of prostacyclin by bovine aortic smooth muscle cells in culture, via 5‐HT2receptors (Coughlin SR, Moskowitz MA, Antoniades HN, Levine L.Proc Natl Acad Sci USA1981;78:7134‐7138). These cells express a synthetic phenotype, whereas the majority of the smooth muscle cells in the media from adult arteries are in a contractile state. We have now compared 5‐HT stimulated prostacyclin production in bovine aortic media explants, a preparation of contractile smooth muscle, with cultured smooth muscle cells derived from the explants. In the 1‐10 &mgr;M range, serotonin stimulates the release of prostacyclin from the explants of bovine aortic media, cultured for a short period. In the presence of cocaine (30 &mgr;M), 1 &mgr;M was sufficient to produce a maximal effect. The stimulatory action of serotonin was sustained with time and did not induce a lasting desensitization. The effect of serotonin on the explants was inhibited only partially (±30%) by ketanserin, a selective and potent 5‐HT2antagonist. It was mimicked by 5‐carboxamido‐tryptamine, a 5‐HT1agonist, but was only weakly inhibited by methiothepin, a 5‐HT1antagonist. As expected, in cultured smooth muscle cells, 5‐carboxamido‐tryptamine was only a weak agonist in stimulating prostacyclin production. In conclusion, it appears that the serotonin effect on prostacyclin production is mediated by different receptors in media explants from bovine aortic media and cultured cells obtained by outgrowth from these explants: a 5‐HT2receptor in the smooth muscle cells in culture and a receptor presenting some similarities with 5‐HT1receptors in the explants. (Circulation Research1989;64:806‐813)