We have investigated the mechanism of action of a series of glutamate derivatives on the cloned excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), expressed in Xenopus laevis oocytes. The compounds were tested as substrates and competitive blockers of the glutamate transporters. A number of compounds showed contrasting mechanisms of action on EAAT1 compared with EAAT2. In particular, (2S,4R)-4-methylglutamate and 4-methylene-glutamate were transported by EAAT1, with Km values of 54 microM and 391 microM, respectively, but potently blocked glutamate transport by EAAT2, with Kb values of 3.4 microM and 39 microM, respectively. Indeed, (2S,4R)-4-methylglutamate is the most potent blocker of EAAT2 yet described. (+/-)-Threo-3-methylglutamate also potently blocked glutamate transport by EAAT2 (Kb = 18 microM), but was inactive on EAAT1 as either a substrate or a blocker at concentrations up to 300 microM. In contrast to (2S,4R)-4-methylglutamate, L-threo-4-hydroxyglutamate was a substrate for both EAAT1 and EAAT2, with Km values of 61 microM and 48 microM, respectively. It seems that the chemical nature and also the orientation of the group at the 4-position of the carbon backbone of glutamate is crucial in determining the pharmacological activity. The conformations of these molecules have been modeled to understand the structural differences between, firstly, compounds that are blockers versus substrates of EAAT2 and, secondly, the pharmacological differences between EAAT1 and EAAT2.