The substance P innervation of the rat parotid was investigated using retrograde tracing with the fluorescent dye True Blue, in combination with the immunohistochemical localization of substance P. Retrogradely-labelled cells were seen in the otic, trigeminal, superior cervical and nodose ganglia. A proportion of the True Blue-labelled cells in the otic and trigeminal ganglia also contained substance P-like immunoreactivity providing direct evidence for a dual origin of substance P in the rat parotid. In addition, these results imply that in the rat otic ganglion substance P and acetylcholine coexist.