Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5- bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5- fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5- iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5- iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)- willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.