Transscleral delivery of triamcinolone acetonide into the vitreous using sub-Tenon's injections may be a safer alternative to reduce the sight-threatening complications of direct intravitreal injections. However, sub-Tenon's injections have demonstrated low and poorly sustained vitreous drug levels in animal studies. To improve our understanding of the clearance mechanisms of corticosteroids, we evaluated vitreous drug levels following sub-Tenon's injection of triamcinolone acetonide in rabbits with selective elimination of conjunctival lymphatic/blood vessels and the choroid. Pigmented rabbits were given a sub-Tenon's injection of a preservative-free triamcinolone acetonide formulation of either a 10- or 20-mg dose in the superotemporal quadrant. The effect eliminating both conjunctival and choroidal clearance was evaluated by injecting the drug, followed by immediate euthanasia, effectively terminating both lymph and blood flow in the conjunctiva and choroid. To inhibit only the clearance from conjunctival lymphatics/blood vessels of a sub-Tenon's injection of triamcinolone acetonide, a group of rabbits had a 'conjunctival window' created by incising an 7 mmx7 mmx7 mm square through the conjunctiva to bare sclera in the superotemporal quadrant. To eliminate only the clearance of drug from the choroidal circulation, cryotherapy was performed in another group of rabbits creating a chorioretinal scar in the superotemporal quadrant. Following the sub-Tenon's drug injection, the eyes were enucleated in all groups after 3 hr and vitreous drug levels were measured with HPLC. In normal animals, a 10-mg sub-Tenon's injection showed no detectable vitreous drug levels; however, a 20-mg injection showed positive vitreous drug levels. This suggested that collectively, the transscleral clearance mechanisms inhibiting delivery into the vitreous may be saturated with a drug depot that has a higher release rate. A 10-mg sub-Tenon's drug depot was able to deliver drug into the vitreous when both the conjunctival and choroidal drug clearance was eliminated by euthanizing the animal immediately following the drug injection. In rabbits that had only a 'conjunctival window', selectively eliminating conjunctival drug clearance, vitreous drug levels were detected. However, in rabbits that had only cryotherapy, selectively eliminating choroidal drug clearance, vitreous drug levels were not detected suggesting that the conjunctival lymphatics/blood vessels may be an important barrier to the transscleral delivery of triamcinolone acetonide. Variability in the vitreous drug levels between rabbits in each group precluded statistical testing. In summary, the rabbit appeared to demonstrate saturable ocular barriers to transscleral delivery of triamcinolone acetonide into the vitreous following a sub-Tenon's injection. The results suggested that the conjunctival lymphatics/blood vessels may be an important barrier to the delivery of triamcinolone acetonide to the vitreous in this rabbit model. The barrier location and clearance abilities of the ocular tissues are important to consider when developing a successful transscleral drug delivery system. Animal models, retaining the dynamics of blood and lymph flow, may improve the basic understanding of the ocular barriers involved with transscleral drug transport and warrants further investigation.