The distribution of 3H and 14C activities in intraocular fluids and tissues was studied after topical application of 3H-prostaglandin F2 alpha (3H-PGF2 alpha) and/or 14C-arachidonic acid (14C-AA) to rabbit eyes; intravenous (i.v.) infusion of 3H-PGF2 alpha into rats; and after incubation of rat or rabbit globes in media containing these and other tracers. Thirty min after topical application of 14C-AA, the order of distribution of 14C per unit of tissue weight was cornea much greater than sclera congruent to ciliary body congruent to iris congruent to aqueous greater than retina-choroid greater than vitreous greater than lens. The distribution of 3H was sclera greater than cornea greater than retina-choroid congruent to ciliary body greater than aqueous greater than vitreous (lens congruent to 0; iris congruent to 0). After i.v. infusion of 14C-sucrose and 3H-PGF2 alpha into rats for 1 to 5 min, the globe/blood ratio of 3H was significantly lower than that of 14C. When isolated rat globes were incubated, the order of tracer uptake into the whole globe was AA greater than thiourea greater than PGF2 alpha greater than sucrose, while the order of entry into the aqueous was thiourea greater than sucrose greater than AA greater than -PGF2 alpha. The isolated rabbit cornea accumulated large amounts of 14C-AA which was not readily elutable and much smaller amounts of 3H-PGF2 alpha which was readily elutable. It is concluded that the sclera is highly permeable to PGF2 alpha, but that the cornea is an effective permeability barrier to this, and presumably all other PGs. The passage of AA through the outer coats of the globe is limited by its chemical incorporation into the cornea, sclera and conjunctiva. Thus, inhibition of the adverse intraocular effects of topically applied AA by topically applied drugs may only reflect the ability of these drugs to inhibit the synthesizing capacity of the superficial layers of the globe rather than that of the anterior uvea and other intraocular tissues.