Nitric oxide synthase (NOS)-containing neurons (localized using NADPH diaphorase histochemistry or NOS immunoreactivity) and vasoactive intestinal peptide-immunoreactive (VIP-IR) neurons were found in the myenteric plexus of the gastrointestinal tract of the amphibian, Bufo marinus. Only limited co-localization of the two substances was observed in nerve cell bodies, about 11% of the NOS-containing neurons were also labelled by VIP-IR and about 37% of VIP-IR nerve cell bodies contained NOS. The relationship between VIP, NOS and SP-IR in nerve fibres in the circular muscle was examined. There was partial co-localization of VIP and NOS, but no co-localization of NOS or VIP with substance P. Of fibres that were immunoreactive for VIP or NOS, fewer than 10% contained VIP alone.