Bladder urothelium acts not only as a barrier to urine but also as a sensory epithelium releasing and responding to numerous chemical transmitters including ATP and acetylcholine (ACh). Many lower urinary tract disorders are associated with a change to this pattern of release and its better characterisation is crucial to develop future treatments. We have investigated pathways of ATP and ACh release, with a novel technique (1) that uses freshly isolated rather than cultured bladder urothelial cells. Male, Dunkin-Hartley guinea-pig bladders were excised, the mucosa removed and incubated with trypsin-EDTA in phosphate-buffered saline, at 37οC for 40 minutes. Urothelial cells were released by gentle titration, washed with Tyrode’s (no added Ca), counted and incubated (>90 mins) in 45 μl aliquots with chemicals to block possible release pathways. Transmitter release was stimulated by pipetting 20 μl of the suspension up and down, 5 (ACh) or 50 (ATP) times. ATP (lucifererin-luciferase assay, Sigma) or ACh (Amplex®Red ACh/ACholinesterase assay kit) was measured in a 20 µl sample. Data are means±SEM. Differences between sets used paired Student’s t-test, with significance at p<0.05. All agents were from Sigma except Amplex®Red, Invitrogen.The role of vesicular transport was tested with N-ethylmaleimide (NEM, 100 µM) which reduced ATP release to 18.0±5.1% of control (p=0.02, n=8) but did not inhibit the more substantial release of ACh (111.4±1.8%, n=5) (Fig 1). Routes of ATP release were tested; i) pannexins with 1 mM probenecid (ATP release reduced to 79.7±7.8%, p=0.012, n=11) or 50 µM carbenoxolone (70.5±6.5%, p=0.009, n=14): ii) connexins with 1.8 mM CaCl2(67.7±5.1% p<0.001, n=30) or flufenamic acid (150µM) which blocks connexins in addition to other ion channels (40.1±5.9 %, p=0.002, n=6). Combining carbenoxolone and calcium was additive (48.2±8.2%, p=0.032, n=7) supporting a role for both pannexin and connexin channels. These findings identify possible targets to modulate urothelial transmitter release.