Concentrations of extracellular glycine in the central nervous system are regulated by Na⁺/Cl⁻-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2by NAGly and by the related compoundsN-arachidonyl- γ-aminobutyric acid and N-arachidonyl-D-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl- L-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg⁵³¹, Lys⁵³², and Ile⁵⁴⁵ that contribute to the differences in NAGly sensitivity.