Nuclei of the medial septum/diagonal band region of the mammalian forebrain contain neurons that give rise to the septohippocampal pathway, which has separate cholinergic and GABAergic components. This pathway is known to influence hippocampal-dependent memory and learning processes, but the precise role of each component is unclear. In this study, we tested the hypothesis that fast-firing, non-bursting medial septum/diagonal band neurons are GABAergic. We used brain slice preparations from young adult guinea-pigs and rats, or from weanling rats, to perform current-clamp recordings from medial septum/diagonal band neurons. Recorded neurons were injected with biocytin for subsequent visualization with fluorescent avidin, and then hybridized with a 35S-labeled riboprobe for glutamate decarboxylase-67 messenger RNA. As a positive control, guinea-pig cerebellar Purkinje cells were labeled and hybridized with the riboprobe. As expected, labeled Purkinje cells were glutamate decarboxylase-67 messenger RNA positive. Slow-firing, cholinergic (choline acetyltransferase-positive) guinea-pig medial septum/diagonal band neurons were glutamate decarboxylase-67 messenger RNA negative. Contrary to our hypothesis, of the guinea-pig neurons, only three of 11 fast-firing neurons were glutamate decarboxylase-67 positive. Of the rat medial septum/diagonal band neurons, three of four were positive for glutamate decarboxylase-67 messenger RNA. These data suggest that fast-firing, non-bursting neurons of the medial septum/diagonal band, as sampled by sharp-electrode intracellular recordings in brain slices, may be a heterogeneous group of neurons, some of which are GABAergic. Together with recent data demonstrating the presence of another GABAergic marker, parvalbumin, in fast-firing septal neurons, we conclude that GABAergic septohippocampal neurons include a population of fast-firing, non-bursting neurons. The influence of these neurons on the hippocampus is likely to occur on a shorter time-scale and over a wider range of firing frequencies as compared to slowly firing cholinergic septohippocampal neurons.