The inactivation of pilocarpine by rabbit serum, rabbit aqueous humor, human serum, human aqueous humor, and human plasma proteins was determined quantitatively by the colorimetric technique of Hestrin. Rabbit serum, rabbit secondary aqueous humor, human serum, and human secondary aqueous humor inactivated 100%, 70%, 40%, and 10% of the pilocarpine, respectively. Rabbit primary aqueous humor, human primary aqueous humor, and human plasma proteins did not inactivate pilocarpine. The effect of various chelating agents on the inactivation of pilocarpine by rabbit serum was measured. Penicillamine, kanamycin sulfate, neomycin sulfate, ethambutol hydrochloride, and triethylenetetramine disulfate, in addition to the previously reported ethylenediaminetetracetic acid (EDTA), prevented rabbit serum from inactivating pilocarpine. Other chelating agents tested had no effect on the reaction. Some aspects of the mechanism of pilocarpine inactivation and possible clinical applications are discussed.