Purpose: To evaluate the reproducibility of the peripapillary retinal nerve fiber layer (RNFL) thickness measurements obtained by Spectralis spectral domain optical coherence tomography (OCT) (Heidelberg Engineering, Heidelberg, Germany) in normal and glaucoma participants. Methods: Participants were recruited from a university-based clinic. Peripapillary RNFL thickness measurements were repeated 3 times during the same visit using the follow-up function. One eye of each participant was randomly selected for statistical analysis. Reproducibility was evaluated using within-subject standard deviation (Sw), coefficient of variation (CV), and intraclass correlation coefficient (ICC). Spearman rank correlation coefficient analyses were used to assess the correlation of the standard deviation of the 3 measurements for each participant with the RNFL thickness value. Results: Forty-five normal participants and 33 glaucoma patients were included in the study. The CVs ranged from 1.45% [overall global (G)] to 2.59% [temporal quadrant (T)] in normal eyes and from 1.74% (G) to 3.22% (T) in the glaucomatous eyes. ICCs ranged from 0.977 (T) to 0.990 (G and inferior-nasal sector) in normal eyes and from 0.983 (T) to 0.997 (inferior quadrant) in glaucomatous eyes. Sw were from 1.34 μm (G) to 2.39 μm (superior-temporal and inferior-temporal sectors) in normal eyes and from 1.14 μm (G) to 2.25 μm (superior-nasal sector) in the glaucomatous eyes. There were no significant correlations between RNFL thickness values and the measurement variability for each participant. Conclusions: Spectralis OCT shows excellent reproducibility for measuring the peripapillary RNFL thickness values in both healthy and glaucoma participants. Copyright © 2011 by Lippincott Williams & Wilkins.