The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst₅R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH₂-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst₅R constructs, and agonist binding was assessed using membrane binding assays with ¹²⁵I-LTT SRIF-28 ligand. Deletion of the NH₂ terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst₁R and sst₄R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind ¹²⁵I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst₅R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (¹²⁵I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III-VII) to the SRIF ligand binding pocket.