Glutamine is present at high concentrations in the extracellular fluid of the brain. It shuttles between glia cells and neurons, and serves as a precursor for both glutamate and γ-amino butyric acid. Direct actions of glutamine at central neurons are, however, not well understood. Here we showed that l-glutamine (0.5–10 mm) evoked a dose-dependent inward transmembrane current in primarily cultured rat hippocampal neurons. Typical responses were outwardly rectifying and had a reversal potential around 0 mV. The current was partially sensitive towards blockers of ionotropic glutamate receptors and was partially carried by activation of N-methyl-d-aspartate receptors. However, cellular responses to l-glutamine showed clear biophysical and pharmacological differences to l-glutamate-evoked currents. Responses were highly specific for l-glutamine and no responses could be evoked by d-glutamine, l-alanine, l-valine, l-leucine and the system-A-specific agonist α-(methylamino)-isobutyric acid. Together, these data indicate that hippocampal neurons can be depolarized by electrogenic effects specific for l-glutamine.