The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an alpha-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.