Substance P- and neuropeptide Y-like immunoreactivity was demonstrated by immunohistochemical methods in mucosal specimens from the middle ear cleft of the guinea pig and rat. Substance P-like immunoreactivity was seen in corresponding specimens of the rabbit. Substance P-like immunoreactivity was also demonstrated in the tympanic nerve of various animal species and man. These specimens also showed weak immunoreactivity for bombesin. The findings suggest the presence of substance P in the tympanic nerve. Theoretically substance P may act in the middle ear cleft mucosa in several ways. One possibility is its putative role in the neurochemical control of the middle ear aeration. Another hypothetical possibility is a role in the neurogenic inflammation after noxious stimulation of the middle ear mucosa.