The effect of reduced tear drainage on the intraocular penetration of topically applied fluorescein was studied with fluorophotometry in healthy eyes. Corneal and aqueous fluorescein concentrations could not be followed for the first hour after application of fluorescein because tear film fluorescense influenced fluorophotometric determinations of fluorescein concentrations in cornea and aqueous for at least 30 min. Insertion of punctal plugs in the upper and lower punctum of one eye caused a significant (P less than 0.025) increase in aqueous fluorescein concentrations 1 to 8 h after application of 20 microliters of a 2% solution of sodium fluorescein in the lower conjunctival sac. The achieved concentrations were almost 4 times as large as in the fellow eye. There was some individual variation, but a marked increase was observed in 9 of 11 subjects. Attempts to reduce tear drainage by compressing the tear sac and/or closing the eye lids for 1 min after application of the eye drop had no significant effect on corneal or aqueous concentrations of fluorescein 1 to 8 h later.