--- - |2+ A Key Point List Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure‐volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation, and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop the decentralized (ex vivo) detrusor smooth muscle (DSM)‐denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium‐derived mediators at the luminal and anti‐luminal aspects of the urothelium during filling. Urinary bladders were excised from C57Bl6/J mice and the DSM was removed by fine scissors dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure‐volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate‐ and lipopolysaccharide‐treated denuded bladders were characterized. The preparation wall contains intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacks the DSM and the serosa. Utility of the model for physiology research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g., ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM‐denuded bladder of Cynomolgus monkeys (Macaca fascicularis). The novel model is superior to current models utilized to study properties of the urothelium (e.g., cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enables direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium‐DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.