The amino-terminal domain containing the ligand binding site of the G protein-coupled metabotropic glutamate receptors (mGluRs) consists of two lobes that close upon agonist binding. In this study, we explored the ligand binding pocket of the Group III mGluR4 receptor subtype using site-directed mutagenesis and radioligand binding. The selection of 16 mutations was guided by a molecular model of mGluR4, which was based on the crystal structure of the mGluR1 receptor. Lysines 74 and 405 are present on lobe I of mGluR4. The mutation of lysine 405 to alanine virtually eliminated the binding of the agonist [(3)H]l-amino-4-phosphonobutyrate ([(3)H]l-AP4). Thus lysine 405, which is conserved in all eight mGluRs, likely represents a fundamental recognition residue for ligand binding to the mGluRs. Single point mutations of lysines 74 or 317, which are not conserved in the mGluRs, to alanine had no effect on agonist affinity, whereas mutation of both residues together caused a loss of ligand binding. Mutation of lysine 74 in mGluR4, or the analogous lysine in mGluR8, to tyrosine (mimicking mGluR1 at this position) produced a large decrease in binding. The reduction in binding is likely due to steric hindrance of the phenolic side chain of tyrosine. The mutation of glutamate 287 to alanine, which is present on lobe II and is not conserved in the mGluR family, caused a loss of [(3)H]l-AP4 binding. We conclude that the determinants of high affinity ligand binding are dispersed across lobes I and II. Our results define a microenvironment within the binding pocket that encompasses several positively charged amino acids that recognize the negatively charged phosphonate group of l-AP4 or the endogenous compound l-serine-O-phosphate.