Single cells or simultaneously recorded cell pairs in the medial septum (MS) vertical limb of the diagonal band of Broca (vDBB) in the urethane-anesthetized rat were initially classified as either theta-on or non-related according to the system of Colom and Bland (1987, Brain Res 422:277-286). Subgroups of these cells were then studied under various test conditions that included electrical stimulation of the nucleus pontis oralis (PO) or posterior hypothalamus (PH), microinfusion of carbachol into the PO or PH, and the microinfusion of atropine sulfate or procaine hydrochloride into the PH. Electrical stimulation of either the PO or PH induced theta (theta) activity in the hippocampal formation (HPC), and electrical stimulation of the PO resulted in a simultaneous increase in the discharge rate of all MS/vDBB theta-on cells tested, compared to the rates recorded during HPC large-amplitude irregular activity (LIA). Five of the MS/vDBB theta-on cells were tested consecutively with electrical stimulation of the PO and PH, and were shown to be activated in a similar manner in either condition. Microinfusion of carbachol into either the PO or PH resulted in the induction of HPC theta field activity and the simultaneous intense activation of all MS/vDBB theta-on cells tested. Following the microinfusion of either atropine sulfate or procaine into the PH, electrical stimulation of the PO failed to induce HPC theta field activity or the concomitant rhythmic discharges of all MS/vDBB phasic theta-on cells tested. Microinfusing procaine into the PH also abolished the coupling between all MS/vDBB cell pairs during HPC theta field activity including that between two cell pairs that were coupled during HPC LIA. The data support the following conclusions: 1) The brainstem HPC synchronizing pathway originating in the pons region ascends to the medial septum via the midline posterior hypothalamic region; 2) the present results taken together with previous work suggest that a major component of the ascending synchronizing pathway, up to and including the hippocampal formation, is cholinergic, cholinoceptive, or both, and the receptors involved are primarily muscarinic; 3) the midline posterior hypothalamic region is an important source of inputs to the medial septum and their major contribution is to provide frequency-coded inputs to the MS/vDBB for relay into the hippocampal formation.