Background: Optical coherence tomography (OCT) is a promising new method of quantifying axon thickness in the retinal nerve fiber layer (RNFL) that has been used predominantly by ophthalmologists to monitor glaucoma. Optical coherence tomography is being considered as a potential outcome measure in multiple sclerosis (MS) clinical trials, but no data exist on the reproducibility of this technique in MS centers. Objective: To determine the reproducibility of OCT measurement of mean RNFL thickness in the undilated eyes of healthy control subjects and patients with MS. Design: Prospective analysis of 4 healthy controls to determine interrater, intrarater, and longitudinal reproducibility. Cross-sectional analysis of 3 cohorts of patients with MS (n=396) and healthy controls (n=153). Setting: Multiple sclerosis clinics at 3 academic medical centers. Patients or Other Participants: Healthy controls and patients with MS. Main Outcome Measure: Thickness of RNFL. Results: We found excellent agreement with respect to interrater (intraclass correlation [ICC], 0.89), intrarater (ICC, 0.98), and intervisit (ICC, 0.91) results. Mean RNFL thickness did not vary significantly among research centers for patients with MS (93, 92, and 90 μm) or among healthy controls (103, 105, and 104 μm) by site. Conclusions: We demonstrate that mean RNFL thickness can be reproducibly measured by trained technicians in an MS center using the OCT-3 model. The RNFL measures from cohorts of age-matched controls and patients with MS from 3 different research centers were remarkably similar. ©2008 American Medical Association. All rights reserved.