Aqueous humor is secreted by the bilayered ciliary epithelium. Solutes and water enter the pigmented ciliary epithelial (PE) cell layer, cross gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, and are released into the aqueous humor. Electrical measurements suggest that heptanol reduces transepithelial ion movement by interrupting PE-NPE communication and that gap junctions may be a regulatory site of aqueous humor formation. Several lines of evidence also suggest that net ciliary epithelial transport is strongly region dependent. Divided rabbit iris-ciliary bodies were incubated in chambers under control and experimental conditions, quick-frozen, cryosectioned and freeze-dried. Elemental intracellular contents of NPE and PE cells were determined by electron probe X-ray microanalysis. With or without heptanol, ouabain produced concentration- and time-dependent changes more markedly in the anterior than posterior epithelium. Without heptanol, there were considerable cell to cell variations in Na gain and K loss. However, contiguous NPE and PE cells displayed similar changes, even when nearby cell pairs were little changed by ouabain in aqueous, stromal or both reservoirs. In contrast, with heptanol present, ouabain added to aqueous or both reservoirs produced much larger changes in NPE than in PE cells. The results indicate that: (1) heptanol indeed interrupts PE-NPE junctions, providing an opportunity for electron microprobe analysis of the sidedness of modification of ciliary epithelial secretion; (2) Na and K undergo faster turnover in the anterior than posterior epithelium; and (3) PE-NPE gap junctions differ from PE-PE and NPE-NPE junctions in permitting ionic equilibration between adjoining ouabain-stressed cells.