Schlemm's canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young's modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young's modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8-15 mm Hg). However, increasing intraocular pressure (22-30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.