The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue’s small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned and freeze-dried for electron-probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na⁺,K⁺-ATPase increases Na and decreases K contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produces smaller changes in anterior PE cells, possibly reflecting higher Na⁺ or K⁺ permeability of anterior NPE cells. Inhibiting Na⁺ entry selectively with amiloride, benzamil or dimethylamiloride reduced anterior effects of ouabain by ~50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl^- secretion. In contrast to ouabain’s actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that: (1) enhanced Na+ reabsorption anteriorly, likely through Na⁺ channels and Na⁺/H⁺ exchange, mediates the regional dependence of ouabain’s actions; and (2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.