A new method for the direct measurement of retinal vessel diameter was developed and the results were compared with those of two computer-assisted semiautomatic microdensitometric methods. The direct measurement method was based on automated detection of vessel edges by processing the one-dimensional retinal image obtained by a linear image sensor set in a fundus camera; results are obtained in real-time. Thirty-eight points on 36 vessels (15 points on arteries and 23 points on veins) of two normal volunteers (four eyes) were selected as the measurement points. Two observers measured the vessel diameters at these points and the interobserver variation of the direct method and the microdensitometric methods was compared. The coefficient of variation and the interobserver variation of the direct method for all measurements were 1.71 + 1.13% and 2.25 + 1.92%, respectively. There was no significant difference between the measured values for the two observers with the direct method (paired t-test, P > 0.05), and the interobserver variation of the direct method was smaller than those of the microdensitometric methods. This newly developed direct method for measurement of retinal vessel diameter not only avoids systematic errors that result from film development or the characteristics of the film, but also generates reproducible results in real-time and small interobserver variation.