Antibiotics which are sparingly lipid-soluble, such as the penicillins, cephalosporins and aminoglycosides, penetrate the eye with great difficulty, achieving intravitreal concentrations which are only a small percentage of the peak serum level. As a result, it is common practice to administer these agents by periocular injection. We have compared the penetration of gentamicin in albino rabbits with that in squirrel monkeys, in normal and infected eyes, after retrobulbar and subconjunctival administration. Generally speaking, the subconjunctival route produced levels equal to or higher than those resulting from retrobulbar injection in both species; it was distinctly superior with regard to cornea and aqueous humor. Inflammation had a neutral or enhancing influence upon intraocular penetration in the monkey, but had a more variable effect in the rabbit; this may be related to the balance between diminution of the blood-ocular barrier and increased dissipation of antibiotic into the systemic circulation. Interspecies differences were much more striking in normal than in infected eyes. This underlines the necessity for the investigator to study inflamed eyes if the results are to be relevant to the clinical situation. Iris and choroid-retina from pigmented rabbits contained significantly more clindamycin and less gentamycin and less gentamicin, in terms of diffusible drug, than tissues from albino animals. These discrepancies reflect opposite interactions between melanin and the two antibiotics in vivo.