The neurotransmitter glutamate is recycled through an astrocytic-neuronal glutamate-glutamine cycle in which synaptic glutamate is taken up by astrocytes, metabolized to glutamine, and transferred to neurons for conversion back to glutamate and subsequent release. The extent to which neuronal glutamate release is dependent upon this pathway remains unclear. Here we provide electrophysiological and biochemical evidence that in acutely disinhibited rat neocortical slices, robust release of glutamate during sustained epileptiform activity requires that neurons be provided a continuous source of glutamine. We demonstrate that the uptake of glutamine into neurons for synthesis of glutamate destined for synaptic release is not strongly dependent on the system A transporters, but requires another unidentified glutamine transporter or transporters. Finally, we find that the attenuation of network activity through inhibition of neuronal glutamine transport is associated with reduced frequency and amplitude of spontaneous events detected at the single-cell level. These results indicate that availability of glutamine influences neuronal release of glutamate during periods of intense network activity.