Background: Radiation-induced bladder toxicity (RIBT) is an unavoidable consequence of pelvic radiation therapy leading to bladder dysfunction (1, 2). RIBT is due to direct irradiation of cells within the radiation field and also indirectly due to effects in non-irradiated, bystander cells which respond to signals released by irradiated cells (3). The current study aims to investigate the underlying molecular mechanisms in bystander urothelial cells that contribute to RIBT. Methods: Immortalized human urothelial cells (SV-HUC) were cultured to subconfluence before being irradiated (2Gy) to generate conditioned medium (CM). Non-irradiated SV-HUC were exposed to CM for 1 hour and subsequently studied with qPCR, Western blotting, cell survival assays, and patch-clamping. Data sets were analysed with student’s t-test and one-way analysis of variance, and results were presented as mean±S.E.M with P<0.05 considered as significant. Results: Application of CM to naïve cells decreased clonogenic cell survival (n=3, P<0.05) indicating the possibility of cell death. Protein analysis from these cells by Western blot showed enhanced expression of cleaved caspase-8, cleaved caspase-3 and cleaved PARP (n=3) consistent with activation of pro-apoptotic signalling. These cells also exhibited enhanced protein expression of cleaved caspase-1 and IL-1β, indicating activation and production of pro-inflammatory mediators. Urothelial cells released ATP into the medium, greater than basal levels after irradiation (luciferin-luciferase assays n=3, P<0.05). The possibility that ATP in the CM modulated induction of pro-apoptotic and pro-inflammatory signalling was confirmed by dose-dependent decrease in cell survival (n=3, P<0.05) by ATP and increased expression of pro-apoptotic and pro-inflammatory proteins (n=5, P<0.05) by ATP. Application of ATP (1mM) evoked inward currents when cells were held at -60mV (n=3) indicating the involvement of membrane purinergic receptors/channels. Protein analysis of the purinergic receptor P2X7R, showed its enhanced expression by CM (n=3). Pre-treatment with P2X7R inhibitors A438079 (100µM) or AZ11645373 (0.5µM) attenuated the CM-evoked and ATP-evoked expression/activation of pro-apoptotic caspases-8, 3 and pro-inflammatory caspase-1 resulting in normalisation of cleaved PARP and IL-1β protein levels, respectively (n=3). Transient knockdown of P2X7R mimicked the effects of inhibitors, confirming the pathological role of P2X7R-mediated signalling after irradiation (n=2). Conclusion: Urothelial cells respond to CM from irradiated cells with induction of pro-apoptotic and pro-inflammatory signalling. Our data indicates that ATP/P2X7R signalling contributes to this aspect of RIBT. Targeting P2X7R may therefore present a promising therapeutic strategy for treatment of RIBT.