Purpose. To assess the effects of Rho-associated kinase (ROCK) inhibition on the intraocular penetration of timolol maleate. Methods. Ex vivo porcine corneal penetration of timolol maleate, sotalol hydrochloride, or brinzolamide incubated with or without Y-27632 was determined in vertical Franz diffusion cells. The effect of ROCK inhibition on the vasodilation of porcine conjunctival vasculature was assessed by scanning electron microscopy (SEM) and immunohistochemical staining with subsequent laser-scanning confocal microscopy (LSCM). Experiments were conducted in New Zealand White (NZW) rabbits to assess the effect of ROCK inhibition on the intraocular distribution of timolol maleate. Results. ROCK inhibition resulted in minimal alteration of ex vivo porcine corneal drug penetration of timolol, sotalol, or brinzolamide. SEM and LSCM experiments conducted with conjunctiva and sclera tissue in Franz diffusion cells suggested vasodilation in the conjunctival vasculature in the presence of Y-27632. Pretreatment of the eyes of NZW rabbits with Y-27632 resulted in aggregate fold reductions (1 hour, 0.25-fold; 4 hours, 0.45-fold) of timolol maleate drug concentrations in intraocular tissues (aqueous humor, lens, and iris) versus eyes not receiving Y-27632 pretreatment. Pretreatment with a vasoconstrictor, phenylephrine, resulted in a reversal of the effect of Y-27632 on diminished timolol maleate intraocular penetration in NZW rabbits. Conclusions. ROCK inhibition reduced the intraocular penetration of administered timolol maleate presumably due to increased systemic elimination through the conjunctival vasculature. It is anticipated that care in order and timing of ROCK inhibitor administration will be warranted for those patients who may be on a multiple topical drug regimen for primary open-angle glaucoma. © 2013 The Association for Research in Vision and Ophthalmology, Inc.