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To evaluate the reproducibility of retinal nerve fiber layer (RNFL) thickness measurements in normal and glaucoma patients using the spectral optical coherence tomography (OCT)/scanning laser ophthalmosope (SLO).One experienced operator measured peripapillary RNFL thickness 3 times in succession in 98 normal participants and 79 glaucoma patients by using the test-retest function of the spectral OCT/SLO. The intraclass correlation coefficient (ICC), coefficient of variation, and test-retest variability (TRV) were analyzed for the mean RNFL thickness, and the RNFL thickness in each quadrant and at each clock-hour, in randomly selected eyes. The correlation coefficient was calculated between the TRV and the mean RNFL thickness, and between the TRV and the mean deviation of the Humphrey visual field.The ICC for mean RNFL thickness was 0.988 in the normal group and 0.993 in the glaucoma group. The ICC for RNFL thickness in all quadrants and clock-hours in both normal and glaucoma patients ranged from 0.951 to 0.992. The coefficient of variation was between 1.9% and 5.0% in the normal group and between 2.0% and 5.3% in the glaucoma group. The TRV was between 4.3 and 11.5 μm in the normal group and between 3.8 and 10.0 μm in the glaucoma group. The TRV showed no correlation with the mean RNFL thickness or the mean deviation (Pearson r>0.15, P>0.2).Spectral OCT/SLO equipped with a test-retest function showed excellent reproducibility with regard to RNFL thickness measurements. The severity of glaucoma did not affect the reproducibility. These findings suggest that spectral OCT/SLO fulfills one of the basic premises for the diagnosis and progression analysis of glaucoma.