Plasma membrane-associated glutamate transporters play a key role in signaling by the major excitatory neurotransmitter glutamate. Uphill glutamate uptake into cells is energetically driven by coupling to co-transport of three Na(+) ions. In exchange, one K(+) ion is counter-transported. Currently accepted transport mechanisms assume that Na(+) and K(+) effects are exclusive, resulting from competition of these cations at the binding level. Here, we used electrophysiological analysis to test the effects of K(+) and Na(+) on neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1, the rat homologue of human excitatory amino-acid transporter 3 [EAAT3]). Unexpectedly, extracellular K(+) application to EAAC1 induced anion current, but only in the presence of Na(+) This result could be explained with a K(+)/Na(+) co-binding state, in which the two cations simultaneously bind to the transporter. We obtained further evidence for this co-binding state, and its anion conductance, by analyzing transient currents when Na(+) was exchanged for K(+), and effects of the [K(+)]/[Na(+)] ratio on glutamate affinity. Interestingly, we observed the K(+)/Na(+) co-binding state not only in EAAC1, but also in the subtypes EAAT1 and 2, which, unlike EAAC1, conducted anions in response to K+ only. We incorporated these experimental findings in a revised transport mechanism, including the K(+)/Na(+) co-binding state, and the ability of K(+) to activate anion current. Overall, these results suggest that differentiation between Na(+) and K(+) does not occur at the binding level, but is conferred by coupling of cation binding to conformational changes. These findings have implications also for other exchangers.