We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of δ and κ opioid receptors (δOR and κOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [ 3H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [3H] diprenorphine binding to eight δOR and eight κOR mutants. All mutants except δOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of δOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of κOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions, (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in κOR but are not in δOR. Thus, TM1 may be closer to TM7 in δOR than in κOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in δOR and at 7.34(311) in κOR, suggesting that TM7 in δOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs. © 2005 American Chemical Society.