AbstractFive sites (5HT1A, 5HT1B, 5HT1C, 5HT1D, and 5HT1E) have been detected using 3H‐5HT radioligand methodology and mammalian brain tissue. We report herein that high‐affinity 3H‐5HT binding to homogenates of bovine and rat caudate and cortical tissues cannot be interpreted without including at least one additional 5HT receptor. Competition studies were performed in rat frontal cortex tissues, using pharmacological blockade of 5HT1A, 5HT1B, and 5HT1C receptors to facilitate examination of other possible sites. Ergotamine, under these conditions, produced a low‐affinity competition K1 = 1.2 1μM with a Hill coefficient of 1.0. 5‐carboxyamidotryptamine (5CT), under these conditions, produced a complex competition curve, with an overall K1 value of 101 nM and a Hill coefficient of 0.55. Computer‐assisted analysis of the 5CT competition curve revealed a high‐affinity site (K1 = 16nM) and a low‐affinity site (K1 = 802 nM). Therefore, competition curves produced using ergotamine and 5‐CT indicate that there is a site in rat frontal cortical tissue with high affinity for 5HT, 5CT, and low affinity for ergotamine as well as a site with high affinity for 5HT and low affinity for 5CT and ergotamine. The site with high affinity for 5HT, and low affinity for 5CT and ergotamine resembles the 5HT1E receptor, insofar as these three drugs are concerned. There is no reported site with high affinity for 5HT and 5CT and low affinity for ergotamine (heretofore referred to as 5HT1F). The lack of a high‐affinity ergotamine competition for 3H‐5HT binding to non‐5HT1A, 5HT1B, and 5HT1C sites indicates that there is no detectable 5HT1D binding in this tissue. In rat striatal tissue both 5CT and ergotamine produced complex competition curves with Hill coefficients less than unity (0.6 and 0.65, respectively). Computer‐assisted analysis indicated that a minority of sites (approximately 35%) displayed high affinity for both ergotamine and 5CT, while 65% of the sites displayed low affinity for both 5CT and ergotamine. The site labeled with 3H‐5HT displaying high affinity for 5CT and ergotamine coincides with the pharmacological profile of a 5HT1D site. The site labeled with 3H‐5HT displaying low affinity for 5CT and ergotamine coincides with the pharmacological profile of the 5HT1E site. Results from bovine cortical and striatal tissues reinforce the conclusions drawn from the rat brain data. In summary, detailed studies of specific 3H‐5HT binding to non 5HT1A‐1C sites indicate that (1) there are detectable 5HT1D sites in rat striatal tissue but not in rat cortical tissue, (2) there are detectable 5HT1E sites in rat brain tissue, and (c) a novel high‐affinity 3H‐5HT binding site possessing high affinity for 5CT and low affinity for ergotamine has been detected in rat and bovine brain tissues. © 1992 Wiley‐Liss, Inc.