The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMSs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21 consecutive residues in the fourth TMS (TM4). Eleven of these mutants reacted with charged sulfhydryl-specific reagents, and bound antagonist protected nine of these from reaction. For the mutants in which cysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had no effect on binding, consistent with these residues being inaccessible and with the low-resolution structure of the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Although hydrophobicity analysis positions the C-terminus of TM4 at 4.64, Pro-Pro and Pro-X-Pro motifs, which are known to disrupt alpha-helices, occur at position 4.59 in a number of homologous GPCRs. The SCAM data were consistent with a C-terminus at 4.58, but it is also possible that the alpha-helix extends one additional turn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degree of sequence variation between 4.59 and 4.68 is more characteristic of a loop domain than a helical segment. This region is shown here to be very conserved within functionally related receptors, suggesting an important functional role for this putative nonhelical domain. This inference is supported by observed ligand-specific effects of mutations in this region and by the predicted spatial proximity of this segment to known ligand binding sites in other TMs.