The modulation of the septohippocampal cholinergic pathway by glutamatergic or GABAergic inputs was studied by monitoring the outflow of ACh collected via a transversal microdialysis probe implanted into the hippocampus and other brain areas of freely moving rats. In one set of experiments a transversal microdialysis membrane was inserted in the dorsal hippocampus, drugs were administered intracerebroventricularly through a cannula implanted in the lateral ventricle, and ACh outflow in the dialysate was measured by an HPLC method with an electrochemical detector. The dialysis membrane was usually perfused with Ringer's solution containing 7 microM physostigmine sulfate. Intracerebroventricular injections of the NMDA antagonists 3-((RS)-2- carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 1–50 nmol), MK801 (0.5–20 nmol), and D(-)-2-amino-7-phosphonoheptanoic acid (100 nmol) brought about an increase in hippocampal ACh outflow while the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (0.25–20 nmol) was without effect. The increase in ACh outflow following CPP administration was dose dependent and reached a maximum of about 500%. It was abolished by TTX (0.5 microM) delivered locally to the hippocampus via the dialysis membrane and prevented by intracerebroventricular injection of the GABA agonist muscimol (5 nmol). In a second set of experiments, one microdialysis membrane was inserted in the dorsal hippocampus to detect ACh outflow and another in the septum to administer drugs locally and at the same time detect septal GABA outflow. The septal dialysis membrane was perfused with Ringer's solution without physostigmine, and GABA levels in the dialysate were measured by an HPLC method with a fluorescence detector. CPP (100 microM) perfused through the septum resulted in a decrease in septal GABA outflow and a concomitant increase in hippocampal ACh outflow. Muscimol (100 microM) administration into the septum abolished the effect of CPP on hippocampal ACh outflow but did not affect septal GABA outflow. These results demonstrate that in the septum NMDA receptors tonically activate GABAergic neurons which in turn inhibit the cholinergic septohippocampal neurons.