AIM: To study the ocular bioavailability of a triple dose, single application of sodium fluorescein to the human anterior segment from a novel drug delivery device. METHODS: In a randomised, open label study 22 healthy volunteers applied a single lyophilisate to one eye (+1 minute) and three conventional eye drops (+1, 16, 31 minutes) of fluorescein ophthalmic solution to the fellow eye. The fluorescein dose of the lyophilisate was 204 mg corresponding to three conventional, preservative-free eye drops of 40 ml fluorescein SE Thilo 0.17% (68 micro g each) (Alcon). Fluorophotometry was performed (Fluorotron Master II Ocumetrics, USA) before and +15, 30, 45, 60, 120, 180, 240, 300, 360, 420 minutes after application. The fluorescein concentrations of the corneal stroma and mid-anterior chamber were analysed by paired t test. RESULTS: Cornea and anterior chamber mean values (ng/ml) were significantly higher (p<0.018, paired t test) in the lyophilisate group up to 7 hours after application with the exception of +45 minutes. The mean fluorescein bioavailability from the lyophilisate was up to 11 times higher in the cornea and up to 8.7 times higher in the anterior chamber compared with the three preservative-free eye drops. CONCLUSION: A triple dose was delivered to the human eye with a single lyophilisate application for the first time. A significantly better bioavailability was achieved in the cornea and anterior chamber for up to 7 hours by means of drug application with lyophilisates. The application of medications by means of the lyophilisate will improve the treatment of, for example, glaucoma, bacterial, viral and fungal infections, as well as dry eye syndrome.