PURPOSE. To introduce a three-dimensional (3-D) histomorphometric strategy for characterizing the connective tissue (laminar) and prelaminar neural tissue (prelaminar) components of optic nerve head (ONH) cupping in one bilaterally normal monkey and three monkeys with early experimental glaucoma (EG) in one eye. METHODS. Trephined ONH and peripapillary sclera from both eyes of four monkeys were serially sectioned at either 3-μm thickness (three EG monkeys) or 1.5-μm thickness (the bilaterally normal monkey) with the embedded tissue block face stained and imaged after each cut. Digital section images were aligned and stacked to create a 3-D reconstruction of each ONH. Within 40 digital radial sagittal sections of each reconstruction, Bruch's membrane opening (BMO), the neural canal wall, and the anterior laminar surface were delineated by two delineators. The 80 BMO points were used to establish a BMO-zero reference plane. The parameters prelaminar tissue volume, post-BMO cup (the estimate of the clinical cup), and post-BMO total prelaminar volume (a global measure of ONH connective tissue deformation) were calculated overall and within 15° radial regions. The parameter prelaminar tissue thickness was calculated at each delineated anterior laminar surface point. For each monkey, an intra-animal difference map was generated for each parameter. Overall volume and thickness data were compared between normal and EG eyes by analysis of variance (ANOVA). RESULTS. Regionally variable expansion of post-BMO cup volume and post-BMO total prelaminar volume were present in all three EG eyes and far exceeded the intra-animal, physiologic differences for these parameters in the bilaterally normal monkey. Prelaminar tissue thickness was increased in all three EG monkeys, with the greatest effects present within the peripheral regions of the canal. CONCLUSIONS. These data suggest that in young adult monkeys with more compliant connective tissues, clinical cupping in early glaucoma is primarily due to fixed deformation of the ONH connective tissues and occurs in the setting of prelaminar tissues that are thickened rather than thinned. Copyright © Association for Research in Vision and Ophthalmology.