Abstract Carbonic anhydrase inhibitors (CAIs) lower intraocular pressure by reducing aqueous flow. It has been thought that this pharmacologic reduction of aqueous flow is mediated by the ciliary epithelium, but it is not known whether this cellular action is effected by inhibition of the membranal (CA IV) and/or cytosolic (CA II) carbonic anhydrases of the ciliary epithelium. The isolated ciliary epithelial bilayer maintains its anatomic and functional polarity and generates a transepithelial potential difference (TEP) in an Ussing type chamber. Depletion of HCO 3 -, accomplished either with an HCO 3 --free solution bathing the epithelial bilayer, or, with addition of freely permeant CAIs to HCO 3 --containing media, (from either the PE or NPE side of the bilayer) depolarizes the preparation. Addition of CAIs to an HCO 3 --depleted preparation has no further effect, indicating the specific action of the CAIs. The CAI, 2-p-NH 2benzenesulfonamido-1,3,4,-thiadiazole-5-SO2NH 2, linked to polybutadiene maleic acid yields an impermeant polymer of 20000Da with no loss of activity. At 45μ mthis impermeant polymer caused a 60% increase in the SCC, seen only when the compound was applied to the NPE side of the bilayer. This latter result indicates an effect from inhibition of CA IV in the basolateral membranes of the NPE. Thus there are probably two different cellular actions of CAIs upon the ciliary epithelium to reduce aqueous inflow, cytoplasmic and membranal. The action of NPE basolateral membranal CA IV is probably linked to the chloride/bicarbonate exchanger.