Stimulation of basal forebrain neurons elicits regional cerebral blood flow increases which are reportedly mediated by acetylcholine and nitric oxide. However, the modality of interaction between these two mediators remains unclear. Particularly, little is known about the source, i.e. endothelial, glial and/or neuronal, of the potent gaseous vasodilator nitric oxide. In the present study, we examined, by double immunocytochemical labelling of nitric oxide synthase and choline acteyltransferase at the light and electron microscopic level, the existence of morphological relationships between cortical nitric oxide synthase-containing neurons and cholinergic cells or nerve fibres. Using anterograde tract tracing and selective basal forebrain lesions, we further investigated the origin of the cholinergic input to cortical nitric oxide synthase neurons. The results confirm that cortical nitric oxide synthase-immunoreactive neurons are often associated with the local microvascular bed, show that intracortical neurons immunostained for nitric oxide synthase and choline acetyltransferase belong to two distinct neuronal populations and, further, that a subset of nitric oxide synthase-containing cell bodies and their proximal dendrites receive a cholinergic input which originates primarily from basalocortical projections. Altogether, these findings suggest that cholinergic basal forebrain neurons could increase cortical blood flow partly via a local nitric oxide relay neuron whereby the freely diffusing gas would be the direct smooth muscle vasodilator agent. It is concluded that this interaction might contribute to the complex relationships between the basal forebrain and the cortical microcirculation, interactions which result in fine regulation of cortical perfusion.