The rational design of prodrugs to improve the therapeutic efficacy of existing drugs would be expedited if the nature of the in vivo enzymatic conditions that regenerate the drugs from their prodrugs is known. Using albino and pigmented rabbits as models, this research seeks to delineate the esterase activities in their corneas, irises, ciliary bodies, and aqueous humor, which are intimately involved in the disposition of drugs from topical dosing. This was achieved by monitoring the hydrolysis kinetics of alpha-naphthyl acetate, both in the presence and absence of esterase inhibitors, upon incubation with aqueous humor and homogenates of cornea, iris, and ciliary body. It was found that in both breeds of rabbits, esterase activity was the highest in the iris-ciliary body followed by the cornea and then the aqueous humor, and that multiple esterases probably existed in the aqueous humor, and the ocular tissues studied. However, the esterase activity in the cornea and iris-ciliary body of the pigmented rabbit was greater when compared with the albino rabbit. Based on these results, drugs and prodrugs containing ester linkages can undergo varying extents of esterase-mediated hydrolysis while permeating the cornea and upon entering the aqueous humor, iris, and ciliary body. Moreover, in view of the differences in esterase activity that exist between the albino and pigmented rabbits, it would be necessary to employ both breeds of rabbits in evaluating the rate and extent to which ocular ester prodrugs would be converted to their parent compounds.