The distribution and origin of nerve fibers of presumed sensory nature in the ear drum and middle-ear mucosa of the rat were studied by a retrograde tracing technique in combination with immunocytochemistry. Application of True Blue (TB) on the ear drum or on the middle-ear mucosa labeled nerve cell bodies in the jugular, trigeminal, geniculate and cervical dorsal root ganglia (C2-C4). Judging from the number of TB-labeled nerve cell bodies the jugular and trigeminal ganglia contributed the major component to the sensory innervation of the ear drum and the middle-ear mucosa, while the contribution from the geniculate and cervical dorsal root ganglia was relatively minor. The majority of the TB-labeled nerve cell bodies contained calcitonin gene-related peptide (CGRP), whereas minor populations stored substance P (SP) and neurokinin A (NKA). Nerve fibers containing SP, NKA and CGRP were moderate in number in the middle-ear mucosa and few in the ear drum. Double immunostaining revealed that SP invariably coexisted with NKA in nerve cell bodies in the ganglia examined. The SP/NKA-containing nerve cell bodies constituted a subpopulation of those storing CGRP. The findings indicate that several ganglia project to the ear drum and middle-ear mucosa and that many neuropeptides are involved in the mediation of middle-ear sensitivity.