The sympathetic innervation of the rat cornea was studied by using the method of intraaxonal transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA). In the first set of experiments, the relative number of superior cervical ganglion neurons that innervate the rat central cornea was estimated by the method of retrograde HRP-WGA transport. Following tracer application to the scarificed central corneal surface, 49-198 labeled neurons were observed in the ophthalmic region of the ipsilateral trigeminal ganglion and zero to four cells in the rostral pole of the ipsilateral superior cervical ganglion. In the second set of experiments, the three-dimensional distribution and termination sites of the corneal sympathetic nerve fibers were investigated by the technique of HRP-WGA anterograde transport from the superior cervical ganglion. HRP-WGA-labeled axons in corneal whole mounts were identified by the presence within their axoplasm of linear arrays of HRP-TMB reaction product, and their distribution was plotted faithfully onto line drawings made with a drawing tube attachment. Large numbers of HRP-labeled fibers were found in all animals within the corneoscleral limbus where the majority were associated with blood vessels. Fewer fibers (zero to 14 per animal) entered the cornea proper. The latter fibers entered the peripheral cornea in the deep to middle layers of the stroma and ascended into progressively more superficial layers as they coursed centrally. The majority of fibers branched infrequently in the peripheral cornea and increased in branching complexity near the central cornea. HRP-labeled axonal varicosities suggestive of terminal and preterminal expansions were located preferentially in the subepithelial layer of the corneal stroma and in the basal epithelium. Approximately 75% of the axonal varicosities were located in the central half of the cornea. The results of the current investigation reveal that the rat cornea is innervated sparsely by sympathetic nerve fibers derived from the superior cervical ganglion. These data provide additional support to current theories that corneal sympathetic nerve fibers may influence select aspects of corneal physiology, including ion transport and hydration, mitogenesis and wound healing, and sensitivity.