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To assess the reproducibility of retinal nerve fiber layer thickness and macular thickness measurements using OCT-3.Randomly chosen eyes of healthy individuals were scanned following pupillary dilation by two trained operators (RGO, RV) using OCT-3 (software version A1.1, Carl Zeiss Meditec, Inc., Dublin, CA), three times on separate days within a one-month period. Fast and regular macula (128 A-scans), and fast and regular RNFL (256 A-scans) scanning protocols were performed. Intra- and interoperator measurement reproducibility was evaluated.Ten eyes of 10 subjects (6 females, 4 males) were enrolled. Mean age was 32 ± 11.2 years (range, 21 to 52 years). Intraoperator reproducibility was high for both macular and RNFL thickness measurements. Mean coefficients of variation (CV) for mean total RNFL thickness measurements ranged from 6.9 ± 6.4% to 8.0 ± 3.5% for operators 1 and 2 in fast and regular RNFL protocols. Mean CV for mean macular thickness measurements ranged from 4.7 ± 2.6% to 6.4 ± 5.5% for operator 1 and 2 in fast and regular macula protocols. There was no difference in mean total RNFL and mean foveal thickness measurements performed on different days (P > 0.05 for all measurements in all protocols, for operators 1 and 2, ANOVA). Interop-erator reproducibility was high for both macular and RNFL thickness measurements (P > 0.05 for all measurements in all protocols, paired t test).OCT-3 RNFL and macular thickness measurements are reproducible in normal eyes. These results should be validated in ocular hypertensive and glaucomatous eyes.