Gentamicin sulfate, a new antibiotic effective against Pseudomonas aeruginosa, was administered intramuscularly or subconjunctivally to New Zealand rabbits and drug concentration determined in serum, primary, and secondary aqueous humor by a plate diffusion method. Subconjunctival injection of this drug was well tolerated and produced levels in primary and secondary aqueous humor sufficient to inhibit most sensitive gram-negative organisms. Insignificant levels of gentamicin were found in primary aqueous humor following intramuscular administration of this drug, although secondary aqueous had levels comparable to those seen following subconjunctival administration. These animal data suggest that this drug may be of value in the treatment of spontaneous and perhaps prevention of surgical ocular infections.