Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ∼30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.