Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helical bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His-ProNH2) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts between the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R and proposed a model of the binding pocket of TRH-R [Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]. Here, we provide evidence for two additional direct interactions between TRH and TRH-R. One interaction is between the aromatic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large increase in the half-maximally effective concentration (EC50) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a loss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 of TM-7 and the terminal carboxamide of TRH. Using four direct interactions as anchors, a refined model of the TRH-R binding pocket was constructed using geometry optimization through energy minimization. A novel method for modeling GPCRs based on Monte Carlo and stochastic dynamics simulations is presented in the accompanying paper [Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663].