The effects of unilateral exposure to hypotonic media on the diffusional water permeability of the isolated rabbit conjunctiva were determined. For these experiments, a segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuited conditions. Unidirectional diffusional water fluxes (Jdw) were measured in either direction by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw were determined in control isosmotic conditions and after dilution of one of the bathing solutions from 290 to 108 mOsMolar. This hypotonic condition reduced Jdw by 25-30% (n = 17) when applied basolaterally and by 25% (n = 6) apically. The effects were reversible and were also obtained when the opposite bathing solution contained amphotericin B, selectively permeabilizing the contralateral cell surface. From concomitant changes in transepithelial electrical resistance as well as 14C-mannitol fluxes completed under identical conditions, arguments are presented that the above effect is best explained as a cell regulated reduction in membrane water permeability. Presumably both apical and basolateral membranes can down-regulate their water permeabilities. This response, suggesting a protective mechanism to help maintain cell volume from hypotonicity, was also seen in other studies using the amphibian bladder and the frog cornea, in which the effect was only obtained basolaterally. Thus, regulation of epithelial water permeability appears to be a basic trait common to both amphibians and mammals, although tissue differences exist.