The binding site of the dopamine D2 receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The highresolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligandbinding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, I184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs.T he extracellular loops are important in ligand binding in G protein-coupled receptors (GPCRs) with large molecular weight ligands, such as peptides (1). The role of these loops in aminergic GPCRs that bind small ligands has received much less attention, and it is widely believed that the transmembrane domain (TMD) is sufficient for ligand binding in these receptors (1). In the high-resolution bovine rhodopsin structure, however, the second extracellular loop (E2) folds down into the binding-site crevice to form a lid over retinal (2). It is unknown in aminergic GPCRs whether the E2 structure is similar to that of rhodopsin or whether E2 plays a role in ligand binding.For nearly all rhodopsin-like GPCRs, the disulfide bond between Cys 3.25 [Cys-107 in dopamine D 2 receptor (D2R)] and the conserved Cys in E2 (Cys e2, Cys-182 in D2R) connects E2 with the TMD § (Fig. 1A), and this disulfide bond (SS-E2) is crucial to the structural integrity and function of many GPCRs. The removal of SS-E2 by mutagenesis severely disrupts ligand binding to muscarinic acetylcholine receptors (3, 4) and destabilizes the high-affinity state of the  2 adrenergic receptor (AR) (5). Moreover, antagonist protected the  2 AR from the effects of reduction by DTT (6). Thus, SS-E2 is protected by a conformational change or steric block within the binding site.Although it had been argued that the presence of E2 within the TMD may be a feature unique to rhodopsin (7,8), several reports implicate E2 in ligand specificity in aminergic and other small molecule-ligand GPCRs (reviewed in ref. 9 and Discussion). Our studies of transmembrane segment 4 (TM4) of the D2R also suggested a role for the extracellular end of TM4 and the Nterminal part of E...