The transpiration stream that passes through a plant may follow an apoplastic route, with low resistance to flow, or a cell-to-cell route, in which cellular membranes impede water flow. However, passage of water through membranes can be facilitated by aquaporins thereby decreasing resistance. We investigated the relationship between transpiration, which can be down-regulated by abscisic acid (ABA) or by high humidity, and the osmotic water permeability ( P os ) of protoplasts. By using leaf protoplasts of wild-type (wt) Arabidopsis thaliana plants and of mutants that are low in ABA ( aba1 ) or insensitive to ABA ( abi1 and abi2 ), we found that protoplasts from aba1 and abi mutants have very low P os values compared with those from wt plants when the plants are grown at 45% relative humidity. High values of P os were found 3 h after the addition of ABA to the culture medium of aba1 plants; addition of ABA to abi plants did not restore the P os to wt levels. There was no such increase in P os when excised leaves of aba1 plants were treated with ABA. When the transpiration stream was attenuated by growing the plants at 85% relative humidity, the P os of protoplasts from all plants (wt and mutants) was higher. We suggest that attenuation of the transpiration stream in whole plants is required for the up-regulation of the P os of the membranes, and that this up-regulation, which does not require ABA, is mediated by the activation of aquaporins in the plasma membrane.