The metabotropic glutamate receptors (mGluRs) are key modulators of excitatory neurotransmission in the central nervous system. The eight mGluR subtypes are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain in which the endogenous ligand binding pocket resides. In this study, we have identified four non-conserved amino acid residues that are essential for differentiating mGluR1 from mGluR4. Our approach has been to increase the affinity of the classic mGluR1 agonists, quisqualic acid and ibotenic acid, at mGluR4 by making various point mutations that mimicked mGluR1 residues. Based on ligand docking to homology models, the non-conserved residues, Lys-74, Glu-287, Ser-313, and Lys-317, were chosen for the mutational studies and all of the mutations proved capable of partially or completely restoring the affinities of the ligands. In particular, the mutations K74Y and K317R induced dramatic triple-order-of-magnitude increases in the affinity of ibotenic acid at mGluR4, making the affinity equivalent to that of mGluR1. Furthermore, the affinity of quisqualic acid at mGluR4 was increased to the same level as mGluR1 by the two double mutations, K74Y/K317R and K74Y/E287G. Advanced analysis of ligand conformation and docking procedures were used for the interpretation of these results. The study shows that mGluR subtype selectivity results from a complex interplay of residues shaping the binding pocket, rather than being attributable to a single specific ligand-receptor interaction.