Interstitial cells (ICs) play a role in regulating normal bladder activity. This study explores the possibility that the sub-urothelial and muscle networks of NO/cGMP-responsive ICs are altered in animals with surgically induced outflow obstruction. In sham-operated animals, the urothelium comprised NO-stimulated cGMP-positive (cGMP(+)) umbrella cells, an intermediate layer and a basal layer that stained for nNOS. cGMP(+) sub-urothelial interstitial cells (su-ICs) were found below the urothelium. cGMP(+) cells were also associated with the outer muscle layers: on the serosal surface, on the surface of the muscle bundles and within the muscle bundles. Several differences were noted in tissues from obstructed animals: (1) the number of cGMP(+) umbrella cells and intensity of staining was reduced; (2) the intermediate layer of the urothelium consisted of multiple cell layers; (3) the su-IC layer was increased, with cells dispersed being throughout the lamina propria; (4) cGMP(+) cells were found within the inner muscle layer forming nodes between the muscle bundles; (5) the number of cells forming the muscle coat (serosa) was increased; (6) an extensive network of cGMP(+) cells penetrated the muscle bundles; (7) cGMP(+) cells surrounded the muscle bundles and nodes of ICs were apparent, these nodes being associated with nerve fibres; (8) nerves were found in the lamina propria but rarely associated with the urothelium. Thus, changes occur in the networks of ICs following bladder outflow obstruction. These changes must have functional consequences, some of which are discussed.