We have utilized a guinea pig in vitro brain slice preparation of the medial septum (MS) and the vertical and horizontal limbs of the nucleus of the diagonal band of Broca (nDBB) to identify and classify different cell types within cholinergic nuclei. Utilizing a double-labeling technique which pairs intracellular injection of the fluorescent dye Lucifer yellow with acetylcholinesterase (AChE) histochemistry, we were able to correlate electrophysiological characteristics with a specific cholinergic cell marker. We report that at least two cell groups can be identified electrophysiologically within the MS/nDBB complex, and one population of neurons demonstrates distinct electrophysiological characteristics that are highly correlated with positive AChE-staining.