The distribution of esterases within a cell can influence the rate and extent at which drugs containing ester linkages would be hydrolyzed. The objective of this study was to evaluate the subcellular distribution of esterases in the bovine eye. Ocular tissue homogenates were subjected to differential centrifugation to yield mitochondrial, microsomal and cytoplasmic fractions. Each fraction was then incubated with 1-or 2-naphthyl esters and its esterase activity determined. No esterase activity was detected in the mitochondrial fraction. For the corneal epithelium and iris-ciliary body about 80% of the esterase activity in the tissue homogenates, on a per milligram protein basis, was associated with the microsomes, the remainder was associated with the cytoplasm. Of the tissues studied, the irisciliary body had the highest esterase activity, 15 and 7 times that in the corneal epithelium and corneal stroma, respectively. The corneal endothelium was devoid of esterase activity. In comparison with the rabbit eye, the specific esterase activity in the bovine eye was lower. Surprisingly, unlike the rabbit, the corneal epithelium of the bovine eye was enzymatically less active than the stroma. Based on these preliminary data, it was concluded that the esterase activity varies with the tissue and that within a cell the esterases were distributed between the cytoplasm and the microsomes according to a ratio specific for a tissue.