The biological activity of a number of ophthalmic drugs is influenced by drug-protein interaction in tissues and fluids of the eye. High concentration of protein in lacrimal fluid, in both normal and pathological states, coupled with a relatively rapid turnover of this fluid, which moves drug solution away from the eye, leads to a considerable loss in drug activity for drugs that bind to protein. High levels of protein, as occur in some pathological states, and a slower, but substantial, turnover rate of aqueous humor can also lead to significant drug loss and a decrease in drug activity for compounds that complex with proteins. The present study, utilizing both in vitro and in vico experiments, shows that drug-protein interaction in tissues and fluids of the eye occurs and that this interaction has an enormous influence on drug bioavailability. Equilibrium dialysis experiments, using pilocarpine nitrate, sulfisoxazole, and methylprcdnisolone. demonstrated that extensive binding to proteins in tears, cornea, and aqueous humor does occur. In addition, pupillary diameter experiments, using pilocarpine nitrate as the test agent, illustrated the influence of drug-protein interaction on drug bioavailability. A discussion of drug binding in combination with tear and instilled fluid dynamics is presented. It is suggested that this phenomenon can be responsible, partly or wholly, for some reported anomalous observations associated with drug therapy in the eye.