The present study was undertaken to characterize the pre- and postsynaptic constituents of the basal forebrain (BF) projection to the prefrontal cortex in the rat, and determine whether it includes glutamatergic in addition to established gamma-aminobutyric acid (GABA)ergic and cholinergic elements. BF fibres were labelled by anterograde transport using biotin dextran amine (BDA) and dual-stained for the vesicular transporter proteins (VTPs) for glutamate (VGluT), GABA (VGAT) or acetylcholine (VAChT). Viewed by fluorescence microscopy and estimated by stereology, proportions of BDA-labelled varicosities were found to be stained for VGluT2 (and not VGluT1 or 3), VGAT or VAChT (representing, respectively, approximately 15%, approximately 52% and approximately 19% within the infralimbic cortex). Each type was present in all, though commonly most densely in deep, cortical layers. Material was triple-stained for postsynaptic proteins to examine whether BDA+VTP+ varicosities might form excitatory or inhibitory synapses, respectively, labelled by postsynaptic density-95 kDA (PSD-95) or gephyrin (Geph). Viewed by confocal microscopy, a majority of BDA+/VGluT2+ varicosities were found to be apposed to PSD-95+ elements, and a majority of BDA+/VGAT+ varicosities to be apposed to Geph+ elements. Other series were triple-stained for cell marker proteins to assess whether the varicosities contacted interneurons or pyramidal cells. Viewed by confocal microscopy, BDA-labelled VGluT2+, VGAT+ and VAChT+ BF terminals were all found in contact with calbindin+ interneurons, whereas VGAT+ BF terminals were also seen in contact with parvalbumin+ interneurons and non-phosphorylated neurofilament+ pyramidal cells. Through distinct glutamatergic, GABAergic and cholinergic projections, the BF can thus influence cortical activity in a diverse manner.