Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+), 1 H(+), and the counter-transport of 1 K(+) ion. In addition to coupled ion fluxes, glutamate and Na(+) binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonate-modified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.