The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (Pd) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by "perforating" the apical membrane with amphotericin B. The addition of this antibiotic increased Pd from 1.12 +/- 0.10 x 10(-4) cm/sec (n = 7) to 4.08 +/- 0.33 x 10(-4) cm/sec (n = 7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl2 (10(-3) M) decreased Pd by 52% and parachloromercuribenzoic acid (pCMB) (1.4 x 10(-4) M) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52 +/- 0.23 kcal/mol (n = 3), in the control situation, to 9.99 +/- 0.91 kcal/mol (n = 4) in the presence of 1.4 x 10(-4) M pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders. pCMB (0.5 or 1.4 x 10(-4) M) was found to inhibit water exit by 91 +/- 2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.