The possible co-localization of choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) in the nerve fibers of cat cerebral arteries was examined by a sequential double-labeling immunohistochemical method. Diaminobenzidine and tetramethylbenzidine were used as chromogens to distinguish ChAT (protein) and VIP (peptide) immunoreactivities. Since available fixatives often did not provide simultaneous preservation of optimal protein and peptide immunoreactivities, a new fixative, buffered periodate-paraformaldehyde-picric acid-formaldehyde-lysine (PPPFL), was formulated and tested. PPPFL fixative is more reliable for simultaneously preserving ChAT and VIP immunoreactivities than were periodate-lysine-paraformaldehyde (PLP) fixative, Zamboni's fixative, or 2% paraformaldehyde solution alone. Using PPPFL as fixative, both ChAT immunoreactive (ChAT-I) and VIP-immunoreactive (VIP-I) fibers in cerebral arteries appeared as bundle and fine fibers. Most ChAT-I and VIP-I fibers were separate. Portions of ChAT-I and VIP-I fibers often ran closely in parallel or across each other. Overlaying of VIP-I on ChAT-I fibers and relay connections between them were also observed. These morphological data suggest the potential functional interactions between cholinergic and VIPergic innervations. In less than 5% of the fibers examined did ChAT and VIP immunoreactivities appear to be co-localized. These data therefore do not support the hypothesis that acetylcholine and VIP are co-localized in most fibers innervating the cerebral arterial wall.