We previously reported that insulin accumulated in the retina and optic nerve following ocular application. Since the optic nerve is surrounded by meninges and cerebrospinal fluid (CSF) and since it extends back to the thalamus, we examined whether the topical application of insulin eye drops also resulted in the accumulation of insulin in the CSF and brain. The data presented in this paper show that this is in fact the case. Following the ocular application of a 0.75% solution of porcine insulin, significant concentrations of insulin were demonstrable in the CSF extracted from the cisterna magnum, as well as in three brain regions. While it is not yet clear how insulin got into these target tissues, our data argue against a mechanism involving uptake from the blood (a fraction of topically applied compounds normally enters the vasculature through the conjunctiva and nasal mucosa). It is theorized that insulin may enter the CSF surrounding the optic nerve and by so doing, not only disseminate throughout the CSF space but also throughout the brain. The implications of these findings for central nervous system drug delivery are discussed.