Using a double labeling indirect immunofluorescent technique, we studied the guinea pig trigeminal ganglion and eye for co-localization of substance P and calcitonin gene-related peptide. In the trigeminal ganglion, the number of neurons immunoreactive for calcitonin gene-related peptide significantly outnumber those immunoreactive for substance P, but virtually all substance P positive neurons are immunoreactive for calcitonin gene-related peptide. In the eye, a complex pattern of co-localization is present; both peptides co-localize in most immunoreactive nerve fibers. Nerve fibers immunoreactive only for calcitonin gene-related peptide tend to be concentrated in the cornea and posterior ciliary body. Nerve fibers immunoreactive only for substance P are present in relation to both iris muscles. Sensory denervation by intracranial transection of the ophthalmic and maxillary nerves fails to eliminate these substance P positive but CGRP negative iris nerve fibers. These findings indicate an alternative origin for substance P immunoreactive nerves supplying the iris muscles in this species.