We have previously reported that spatial reference memory (RM) training-induced alterations in hippocampal cholinergic activity as measured by sodium-dependent high-affinity choline uptake (SDHACU). Each training session was found to induce an immediate (30 s) increase in SDHACU followed (30 s to 15 min posttest) by a deactivation and long-lasting inhibition (15 min to 24 h) of this cholinergic marker. The present experiments were designed to assess the role of septal glutamatergic receptors in this posttraining cholinergic deactivation. In the first experiment, the effects of intraseptal injections of different doses of glutamic acid and glutamatergic antagonists (kynurenic acid, KYN, and AP5) on hippocampal SDHACU were studied in awake but otherwise resting (i.e., naive) mice. The results showed that glutamic acid at the lowest dose used (5 ng) produced a decrease in SDHACU, whereas both glutamatergic antagonists produced a dose-related increase in this cholinergic marker. It was concluded that septal glutamatergic receptors mediate a tonic inhibitory input on the cholinergic cells. Hence, in a second experiment the effect of intraseptal injections of KYN (5 ng) on the training-induced changes in hippocampal cholinergic activity were assessed following variable amounts of radial maze RM training. Trained mice were injected 20 min before the first or the ninth training session and killed 30 s or 15 min posttraining for determination of SDHACU. KYN slowed the posttesting cholinergic deactivation (disinhibition), this effect being more marked in good learners than in bad learners. The present findings suggest that septal glutamatergic receptors mediate an inhibitory input on the cholinergic cells, and that this input could play a role in memory consolidation.