In men, testosterone levels decline by 1% per year after the age of 40. Reduced androgen levels may directly contribute to lower urinary tract symptoms and bladder dysfunction, although the mechanisms are unclear. This study examined the effect of low testosterone and testosterone replacement on key mechanisms involved in local bladder function. Intraluminal release of the mediators ATP and ACh in response to bladder distension was measured in whole bladders from rats 8 weeks following castration, whilst bladder contractility was assessed using isolated strips. Human urothelial cells were cultured under low, physiological and supra-physiological testosterone conditions for 24 h or 5 days, and stretch-induced release of ATP and ACh was measured. Phasic contractile activity of bladder strips, agonist-induced reponses to carbachol and isoprenaline and nerve-evoked contractions were unaffected by castration. The acetylcholinesterase inhibitor neostigmine significantly increased amplitude of phasic activity only in bladder strips following castration, and this was prevented by testosterone replacement. Intraluminal ACh release following bladder distension was significantly reduced following castration, whilst ATP release was unaffected. In contrast, stretch-induced ATP release from urothelial cells was significantly enhanced in low testosterone conditions, whilst ACh release was unaltered. Testosterone-replacement to physiological levels prevented these changes. Whilst androgen deficiency of 8 weeks does not directly affect contractility of bladder smooth muscle, urothelial mediator release is sensitive to changes in testosterone. These changes in mediator release may be an early effect of the decline in testosterone and could affect sensory pathways in the longer term, contributing to the urinary symptoms and bladder dysfunction seen in androgen-deficient men.