2-Oxoglutarate (alpha-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na(+)-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, Vmax, and negative cooperatively. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases Vmax. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.