The medial septum contains cholinergic and GABAergic neurons that project to the hippocampal formation. A significant proportion of the septohippocampal neurons (SHN) exhibit a rhythmically bursting (RB) activity that is involved in the generation of the hippocampal theta rhythm. The neurochemical nature of septal RB neurons is not firmly established. To address this question, the septal unit activity has been recorded in rats after selective destruction of the cholinergic septal neurons by the immunotoxin 192 IgG-saporin. Experiments have been performed in urethan-anesthetized and unanesthetized rats, 14-21 days after lesion. Acetylcholinesterase (AChE) histochemistry revealed a near-complete loss of cholinergic septal neurons and of cholinergic fibers in the hippocampus. The recorded neurons were located in the medial septum-diagonal band of Broca area. A number of these neurons were identified as projecting to the hippocampus (SHN) by their antidromic response to the electrical stimulation of the fimbria-fornix. In urethan-anesthetized lesioned rats, the percentage of RB neurons decreased significantly as compared with controls (17 vs. 41% for SHNs and 5 vs. 19% for unidentified septal neurons). The axonal conduction velocity and the burst frequency of the SHNs that retained a RB activity were higher in lesioned as compared with control rats. The number of spikes per burst was lower and the burst duration was shorter in lesioned rats as compared with controls. The urethan-resistant hippocampal theta was altered both in terms of frequency and amplitude. In unanesthetized lesioned rats, no RB septal neurons were found during arousal, as compared with 25% in controls. Their number was also markedly reduced during paradoxical sleep (9.7 vs. 38.5%). Histochemistry in 192 IgG-saporin-treated rats showed that RB neurons were found in areas devoid of AChE-positive neurons but containing parvalbumine-positive (presumably GABAergic) neurons. These data show that RB activity is considerably reduced after selective lesion of the cholinergic medial septal neurons. They suggested that the large majority of the RB septal neurons are cholinergic and that the few neurons that display RB activity in lesioned rats are GABAergic.