The characteristic electroencephalographic patterns within the hippocampus are theta and sharp waves. Septal neurons are believed to play an essential role in the rhythm generation of the theta pattern. The present study examined the physiological consequences of complete and selective damage of septohippocampal cholinergic neurons on hippocampal theta activity in rats. A selective immunotoxin against nerve growth factor receptor bearing cholinergic neurons (192 immunoglobulin G-saporin), [Wiley R. G. et al. (1991) Brain Res. 562, 149-153] was infused into the medial septal area (0.11-0.42 microgram). Hippocampal electrical activity was monitored during trained wheel running, drinking and the paradoxical phase of sleep, as well as following cholinomimetic treatment. A moderate dose of toxin (0.21 microgram) eliminated the septohippocampal cholinergic projection, as evidenced by a near total absence of choline acetyltransferase-immunoreactive neurons in the medial septum and the vertical limb of the diagonal band, and by the absence of acetylcholinesterase-positive fibers in the dorsal hippocampus. In the same rats, parvalbumin immunoreactivity, a reliable marker for septohippocampal GABAergic neurons, [Freund T. F. (1989) Brain Res. 478, 375-381], remained unaltered. In addition, retrograde transport of the tracer fluorogold demonstrated that the parvalbumin cell population preserved its axonal projection to the hippocampus. Following toxin treatment, the power of hippocampal theta, but not its frequency, decreased in a dose-dependent manner. Reduction of theta power occurred between three and seven days after the toxin treatment and remained unaltered thereafter up to eight weeks. A dose which eliminated all septohippocampal cholinergic neurons (0.21 microgram) left a small but significant theta peak in the power spectra during wheel running, paradoxical phase of sleep and intraseptal infusion of carbachol (5 micrograms). Peripheral administration of physostigmine (1 mg/kg) induced only slow (1.5-2.0 Hz) rhythmic waves. No changes were observed in the gamma (50-100 Hz) band. These findings indicate that the integrity of the septohippocampal GABAergic projection is sufficient to maintain some hippocampal theta activity. We hypothesize that cholinergic neurons serve to increase the population phase-locking of septal cells and thereby regulate the magnitude of hippocampal theta.