³H]Serotonin (5‐hydroxytryptamine, [³H]5‐HT) was used as a radioligand probe of brain 5‐HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 μM pindolol (to block 5‐HT_1Aand 5‐HT_1B receptors), and 100 nM mesulergine (to block 5‐HT_1c and 5‐HT₂ receptors). One of these sites demonstrated high affinity for 5‐carboxyamidotryptamine (5‐CT) and er‐gotamine, consistent with the known pharmacology of the 5‐HT_1D receptor; the second site demonstrated low affinity for 5‐CT and ergotamine. Computer‐assisted analyses indicated that both drugs displayed high affinities (K_i values of 1.1 nM and 0.3 nM for 5‐CT and ergotamine, respectively) for 55% of the sites and low affinities (K_i values of 910 nM and 155 nM for 5‐CT and ergotamine, respectively) for 45% of the sites. To investigate the non‐5‐HT_1D component of the binding, 100 nM 5‐CT (to block 5‐HT_1A, 5TTT_1B, and 5‐HT_1D receptors) was coincubated with [³H]5‐HT, membranes, and mesulergine. The remaining [³H]5‐HT binding (hereafter referred to as “5‐HT_1E”) displayed high affinity and saturability (K_D, 5.3 nM; B_max, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5‐CT and ergotamine displayed the highest selectivity for the 5‐HT_1D site versus the 5‐HT_1E site. The interaction of the 5‐HT_1E site with a GTP‐binding protein was demonstrated; the nonhydrolyzable derivatives of GTP, guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS) and 5′‐guanylylimidodiphosphate [Gpp(NH)p], potently inhibited binding of [³H]5‐HT to the 5‐HT_1E site (IC₅₀ values of 16 and 172 nM, respectively) while adenosine 5′‐0‐(3‐thiotriphosphate) (ATPγS) and 5′‐adenylylimidodi‐phosphate [App(NH)p] were without effect. The high affinity of 5‐HT for the site, the unique pharmacological profile of the site, and the interaction of the site with a GTP‐binding protein indicate that this site represents a unique 5‐HT receptor subtype heretofore undescribed, and which we propose to call 5‐HT_1E in keeping with the current system of nomenclature in the 5‐HT receptor field.