PURPOSE. To assess the ability to detect the neural canal opening (NCO) and its characteristics within three-dimensional (3-D) histomorphometric and 3-D spectral domain optical coherence tomography (SD-OCT) reconstructions of the optic nerve head from nonhuman primate (NHP) eyes. METHODS. NCO was delineated within 40 radial, sagittal sections of 3-D histomorphometric reconstructions of 44 normal eyes of 38 NHPs, each perfusion fixed at IOP 10 mm Hg, and 3-D SD-OCT (Spectralis; Heidelberg Engineering, Heidelberg, Germany) volumes acquired in vivo from a separate group of 33 normal eyes of 24 NHPs. Within all reconstructions, a least-squares ellipse was fitted to the 80 NCO points. For each eye, the dimensions and plane error (a gauge of planarity) of the fitted ellipse were calculated. RESULTS. The NCO was successfully delineated within every section of each histomorphometric and SD-OCT reconstruction. Median plane error was similar within histomorphometric and SD-OCT volumes (8 μm, range 4-19, histomorphometry, and 10 μm, range 4-26, SD-OCT) and was small relative to the size of the ellipse. Median histomorphometric ellipse dimensions were 1453 μm (major axis, range 1218-1737) and 1066 μm (minor axis, range 808-1263). Median SD-OCT ellipse dimensions were 1512 μm (major axis, range 1191-1865) and 1060 μm (minor axis, range 772-1248). CONCLUSIONS. The NCO is biologically continuous and relatively planar within all 3-D histomorphometric and SD-OCT reconstructions. These characteristics support its further evaluation as a reference plane for cross-sectional and longitudinal measurement of optic nerve head structures using 3-D SD-OCT. © Association for Research in Vision and Ophthalmology.