The presence and distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibers associated with the guinea pig major cerebral arteries was studied by means of immunohistochemical, histochemical and ultrastructural techniques. Anterior arteries of the circle of Willis received a rich supply of perivascular nerve fibers containing NOS immunoreactivity while posteriorly localized arteries presented a moderate to sparse innervation. A double immunofluorescence staining technique revealed that NOS was localized in nerve fibers distinct from those displaying substance P or tyrosine hydroxylase. Combined immunofluorescence and histochemical staining of the same preparation indicated that NOS immunoreactivity was localized in putative cholinergic nerve fibers (identified by their acetylcholinesterase content) and that NADPH-diaphorase activity (a marker for NOS-containing neurons) was found in nerves which also possessed VIP immunoreactivity. The ultrastructural study revealed that NOS immunoreactivity was present in numerous nerve varicosities at the adventitial-medial border. These results suggest that NO and VIP co-exist in putative parasympathetic nerve fibers supplying the guinea pig cerebral arteries and may be release together in response to nervous stimulation.