AIMS/BACKGROUND: Comparison of the diffusion coefficient through the blood-aqueous barrier of healthy volunteers measured in different cities with identical fluorophotometers using a standardised protocol. METHODS: Healthy volunteers aged between 20 and 70 years were studied in seven European cities. The fluorescein concentration in the anterior segment of each eye was measured with a commercial scanning fluorophotometer 30 and 40 minutes after intravenous fluorescein. The decay of non-protein bound fluorescein concentration in blood plasma was determined with the use of three blood samples taken at 7, 15, and 55 minutes after injection. The diffusion coefficient through the blood-aqueous barrier was calculated from the ratio between the fluorescein concentration in the anterior chamber and the time integral of non-protein bound fluorescein concentration in plasma using specially developed software. RESULTS: The mean values of the diffusion coefficient (SD) (X10(-4) min-1) were 4.76 (1.51) (n = 20, Brussels), 5.48 (2.33) (n = 17, Coimbra), 3.47 (2.09) (n = 12, Cologne), 6.09 (2.77) (n = 21, Frankfurt), 3.85 (1.59) (n = 11, Ghent), 4.99 (1.69) (n = 23, Leiden), and 4.87 (1.05) (n = 20, Madrid). The values between centres were similar (Kruskal-Wallis test p > 0.05) except for Cologne and Frankfurt (p = 0.013). No differences were found when repeating measurements (four centres, interval time 1-8 months, Wilcoxon paired test p > 0.39). CONCLUSION: The diffusion coefficients had similar values and standard deviations. The concerted action demonstrated the usefulness of a standardised protocol.