Firing patterns, action potential characteristics and some active membrane properties of guinea-pig medial septum/diagonal band neurons were studied in an in vitro slice preparation. A comparison was made between several types of cells classified according to either extracellularly recorded (n = 130) or intracellularly recorded (n = 30) electrophysiological characteristics. Using multi-barrel extracellular electrodes, three principal cell types were distinguished: slow rhythmic firing cells (29%), fast rhythmic firing cells (65%) and burst-firing cells (6%). Most slow firing cells could also be distinguished from other cell types by their relatively longer action potential duration and a characteristic cadmium-sensitive "hump" in the repolarization phase of the action potential. These characteristics of slow firing cells matched well with the characteristics of cholinergic, slow afterhyperpolarization cells previously identified with intracellular recordings. The action potential shape, firing rate and firing pattern characteristics of about 60% of extracellularly recorded fast rhythmic firing cells matched those of previously identified non-cholinergic fast afterhyperpolarization cells. The remaining extracellularly recorded, rhythmic firing cells (about 10% of slow firing and 40% of fast firing cells) had a mixture of characteristics which precluded unequivocal classification as to cholinergic or non-cholinergic cell type. Using intracellular recording, the bee venom toxin, apamin, was shown to attenuate the characteristic post spike slow afterhyperpolarization of cholinergic cells and greatly enhanced their firing rate to depolarizing pulses. Apamin often attenuated a smaller and more transient afterhyperpolarization found in identified non-cholinergic cells, but firing rate was increased only slightly. Extracellular recordings from slow and fast rhythmic firing cells in the presence of apamin showed that excitability of slow firing cells was enhanced significantly more than fast firing cells. The apamin data support the hypothesis that extracellularly recorded slow firing cells are cholinergic. We conclude that extracellularly recorded medial septum/diagonal band cells characterized by broad action potentials, slow rhythmic firing under microiontophoresed glutamate and a signature "hump" in the falling phase of the action potential are cholinergic cells. Extracellularly recorded fast rhythmic firing cells with a narrow action potential and no "hump" in the action potential are likely to be non-cholinergic cells. This extracellular electrophysiological "fingerprint" for cholinergic medial septum/diagonal band cells in vitro may now be extended to studies in vivo where controversy remains as to the neurochemical identity of basal forebrain cells involved in control of hippocampal slow rhythmic activity.