We compared the ocular penetration of labeled with radioactive carbon gentamicin in squirrel monkeys after subconjunctival and retrobulbar administration. In both normal and infected (Staphylococcus aureus endophthalmitis) eyes, high concentrations of drug were achieved in the sclera and choroid-retina by both routes, while corneal levels were markedly higher after subconjunctival injection than after retrobulbar injection. Regional variations in concentration were evident in these tissues; the highest levels were clustered about the site of injection. Aqueous humor concentrations were lowest in the group with normal eyes treated by the retrobulbar route; vitreous humor levels were extremely low in normal eyes injected subconjunctivally. These data differ from those in rabbits, especially with regard to penetration of the vitreous humor of normal eyes. Interspecies differences were less marked in inflamed eyes. The two species were similar in demonstrating maximum access to the cornea and aqueous humor with subconjunctival injection, and equivalence of the two routes in penetrating the vitreous humor of the inflamed eyes.