The glyoxylic catecholaminergic histofluorescence method was employed on the mucosa of the rat's eustachian tube (ET) in order to study the sympathetic innervation present. One percent neutral red was used as counterstain. Many noradrenergic fibers were demonstrable around blood vessels, glands and submucosa of the ET, but not in the epithelium. In a group of rats following neurectomy, the superior cervical ganglia (SCG) were removed unilaterally or bilaterally. Changes in sympathetic innervation of the ET were examined 14 days after SCG ganglionectomy. In those animals after unilateral SCG ganglionectomy, no noradrenergic histofluorescence was found in the ipsilateral ET, although some scant fluorescence could be detected in the tube's nasopharyngeal (NP) orifice. However, no noradrenergic histofluorescence could be observed in animals bilateral SCG ganglionectomies. Our results indicate that sympathetic innervation of the ET in the rat originates in the SCG, with some cross-innervation of sympathetic fibers occurring in the tube's NP orifice.