BACKGROUND: In clinical practice, the differentiation of glaucomatous from non-glaucomatous cupping can be difficult, even for experienced observers. The purpose of this study was to evaluate the role of optical coherence tomography (OCT) in differentiating glaucomatous from non-glaucomatous optic nerve cupping in a cross-sectional pilot study. METHODS: Eleven consecutive patients presenting to the Duke Eye Center from September 2007 to July 2008 with non-glaucomatous optic nerve cupping and 12 patients with glaucomatous optic nerve cupping were identified. All patients underwent Stratus® OCT imaging: fast macular map, fast retinal nerve fiber layer (RNFL) 3.4 thickness, and fast optic disc protocols. Automated visual field perimetry was performed on the date of OCT scan in non-glaucomatous cupping patients, and from 0-9 months of scan date in glaucoma patients. Eyes were matched by optic nerve cup-to-disc area ratio; average and mean deviation were calculated for each variable. RESULTS: For a similar average RNFL, patients with non-glaucomatous optic nerve cupping had lower nasal and temporal RNFL thickness, as well as lower macular thickness and volume compared to patients with glaucomatous optic nerve cupping. CONCLUSION: OCT appears to be a useful technology in differentiating glaucomatous from non-glaucomatous optic nerve cupping. The pattern of RNFL loss appears more diffuse in non-glaucomatous optic nerve cupping compared to glaucomatous optic nerve cupping. Future studies with larger sample size and specific neuro-ophthalmic causes of optic nerve cupping may further elucidate the role of OCT in this clinical setting.