3H-glutamate (GLU) uptake was measured in hippocampal synaptosomes from rat brain. Addition of sodium nitroprusside (SNP) (Sodium nitroferricyanide), a generator of nitric oxide (NO), produced a time-, temperature-, and dose-dependent inhibition of 3H-GLU uptake. The inhibition was due to changes in both Kd and Vmax of GLU uptake, and it was at least partially reversible upon washing. Addition of reduced hemoglobin (Hb), a substance that binds NO, prevented the SNP-induced depression of uptake. Potassium ferricyanide, a compound similar to SNP, did not cause a reduction in 3H-GLU uptake. Utilization of another generator of NO, S-nitroso-N-acetylpenicillamine (SNAP), produced similar results as did NO itself. Decreases in uptake were also observed in the striatum and cerebellum. Similar treatments did not consistently affect 3H-norepinephrine (NE) uptake, suggesting some selectivity in the NO effect. Thus, the observed inhibition of 3H-GLU uptake appears to be produced by NO, and it may represent a novel type of transynaptic retrograde regulation of transport. If found in vivo, inhibition of uptake activity could also be involved in the toxic effects of NO, the neurotoxicity of glutamate, and other potential neuronal changes associated with NO such as hippocampal long-term potentiation.