The distribution of substance P (SP) within the nerve fibers of the rat cornea and iris was studied using electron microscopic immunohistochemistry. Numerous SP-immunoreactive fibers were found throughout the corneal and iridal stroma. The most intense staining was found within varicosities containing numerous vesicles. Similar SP-immunoreactive varicosities were found in fibers in close proximity to the iris sphincter and dilator muscles. On account of previous ultrastructural work the SP-containing fibers and varicosities must be assumed to be of trigeminal origin. Additionally, the distribution of SP and calcitonin gene-related peptide (CGRP) in corneal and iridal fibers of rats is similar, suggesting that CGRP and SP are colocalized and may be coreleased from the same varicosities.