PURPOSE: Quantitative assessment of nerve fiber layer (NFL) thickness in normal and glaucomatous eyes, and correlation with conventional measurements of the optic nerve structure and function. METHODS: We studied 59 eyes of 33 subjects by conventional ophthalmologic physical examination, Humphrey 24-2 visual fields, stereoscopic optic nerve head photography, and optical coherence tomography. RESULTS: Nerve fiber layer thickness as measured by optical coherence tomography demonstrated a high degree of correlation with functional status of the optic nerve, as measured by visual field examination (P = .0001). Neither cupping of the optic nerve nor neuroretinal rim area were as strongly associated with visual field loss as was NFL thickness (P = .17 and P = .21, respectively). Cupping correlated with NFL thickness only when the cup was small (cup-to-diameter ratio, 0.1 to 0.3) or large (cup-to-diameter ratio, 0.8 to 1.0) (P = .006); there was no correlation between cupping and NFL thickness otherwise. Nerve fiber layer, especially in the inferior quadrant, was significantly thinner in glaucomatous eyes than in normal eyes (P = .04). Finally, we found a decrease in NFL thickness with aging, even when controlling for factors associated with the diagnosis of glaucoma (P = .03). CONCLUSIONS: Nerve fiber layer thickness can be measured using optical coherence tomography. These measurements provide good structural and functional correlation with known parameters.