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, we previously mapped the residues in the third membrane-spanning segment (M3) that are exposed in the biding site crevice [Javitch et al. (1995) Neuron 14, 825]. We have now mutated, one at a time, 24 consecutive residues in and flanking the fifth membrane-spanning segment (M5) to cysteine and expressed the mutant receptors in HEK 293 cells. Thirteen of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by another reversible dopamine antagonist, sulpiride. Thus, the side chains of these residues are exposed in the binding-site crevice. Of the 13 exposed residues, 10 are consecutive, from Phe189 to Phe198. This pattern of exposure is inconsistent with the expectation that M5, like M3, forms a fixed alpha-helix, one side of which is exposed in the binding-site crevice. The exposed region of M5, which contains the serines likely to bind agonist [Strader et al. (1989) J. Biol. Chem. 264, 13752], might loop out into the lumen of the binding-site crevice and be completely accessible to water and thus to MTSEA. Alternatively, the exposed region of M5 might be embedded in the membrane and also in contact with other membrane-spanning segments. At any instant, only a limited set of residues might be exposed in the binding-site crevice; however, M5 might move rapidly to expose different sets of residues.