PURPOSE. We present spatial retinal nerve fiber layer (RNFL) attenuation coefficient maps for healthy and glaucomatous eyes based on optical coherence tomography (OCT) measurements. Quantitative analyses of differences between healthy and glaucomatous eyes were performed. METHODS. Peripapillary volumetric images of 10 healthy and 8 glaucomatous eyes were acquired by a Spectralis OCT system. Per A-line, the attenuation coefficient of the RNFL was determined based on a method that uses the retinal pigment epithelium as a reference layer. The attenuation coefficient describes the attenuation of light in tissue due to scattering and absorption. En-face maps were constructed and visually inspected. Differences between healthy and glaucomatous eyes were analyzed (Mann-Whitney U test), both globally (average values) and spatially (concentric and per segment). RESULTS. RNFL attenuation coefficient maps of healthy eyes showed relatively high and uniform values. For glaucomatous eyes, the attenuation coefficients were much lower and showed local defects. Normal and glaucomatous average RNFL attenuation coefficients were highly significantly different (P < 0.0001) and fully separable. The RNFL attenuation coefficient decreased with increasing optic nerve head distance for both groups, with highly significant differences for all distances (P < 0.001). The angular dependency showed high superio- and inferiotemporal and low nasal values, with most significant differences superio- and inferiotemporally. CONCLUSIONS. Maps of RNFL attenuation coefficients provide a novel way of assessing the health of the RNFL and are relatively insensitive to imaging artifacts affecting signal intensity. The highly significant difference between normal and glaucomatous eyes suggests using RNFL attenuation coefficient maps as a new clinical tool for diagnosing and monitoring glaucoma. Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.