beta-Alanine and taurine are agonists of the glycine receptor (GlyR) which, at low concentrations, antagonize the action of the principal agonist glycine. We analysed the potency of these ligands on alpha 1 subunits mutated at residue R271. GlyRs formed from alpha 1R271K subunits showed a reduction of beta-alanine and taurine affinities and maximal inducible currents; the mutants alpha 1R271Q and alpha 1R271L associated with human hyperekplexia gave no responses to these ligands. Inhibition of glycine-evoked currents by beta-alanine and taurine, however, was similar for all mutant GlyRs. These data are consistent with the existence of two subdomains within the ligand binding region of the GlyR, an agonistic one, which depends on arginine 271, and an antagonistic subsite, which is not connected to this residue.