In frogs and toads the urinary bladder is very important for the maintenance of water balance due to its ability to store water which can be reabsorbed under the action of arginine-vasotocin (AVT). The usage of isolated bladders as a model for studying the osmotic water permeability (OWP) regulation has a disadvantage which relates to high variability of AVT effect among individuals, some showing insensitivity to the hormone. We hypothesized that the response of the bladder to AVT could depend on the colonization of the mucosal epithelium by Gram-negative bacteria. To test this, paired hemibladders of the frog Rana temporaria were used for measurement of OWP and for analysis of Gram-negative bacteria in the bladder tissue or isolated epithelial cells. Among the 206 frogs studied, 41% were infected by different Enterobacteriaceae, with prevalence of Hafnia alvei and Escherichia coli. In infected bladders the basal level of OWP was unchanged, whereas OWP stimulated by AVT was reduced (non-infected: 2.53 ± 0.13, n = 59, infected: 1.21 ± 0.17 µL min−1 cm−2, n = 38, for the 15 min of AVT action, P < 0.001). In the sample, 100% of hemibladders that responded to AVT very weakly (OWP <0.5 µL min−1 cm−2) had a bacterial infection. Overnight treatment of hemibladders with mucosal lipopolysaccharide E. coli decreased OWP induced by AVT, forskolin, or IBMX lowering basal and stimulated level of cAMP. The data obtained indicate that the frog bladder epithelium could be colonized by Gram-negative bacteria, probably of cloacal origin, leading to reduction of sensitivity to AVT and to impairment of the urinary bladder to provide osmoregulation. J. Exp. Zool. 319A: 487–494, 2013. © 2013 Wiley Periodicals, Inc.