Aims: Previously we showed that ischemia alters bladder smooth muscle contractility in the rabbit. This study investigates the role Of Urothelium and eicosanoid-release in ischemic bladder smooth muscle instability. Materials and Methods: Male New Zealand white rabbits were divided into treated (n = 12) and age-matched control (n = 10) groups. The treated group underwent balloon enclothelial injury of the iliac arteries, and then received 4 weeks of cholesterol diet, followed by 4 weeks of regular diet. The control group received a regular diet for 8 weeks. After 8 weeks, blood flow for both the iliac arteries and the bladder as well as bladder oxygen tension were recorded. In one-half of each ischemic and control bladder, the urothelium,vas removed. Bladder tissues were processed for organ bath and enzyme-immunoassay (EIA) of prostaglandins (PGs) and leukotrienes (LTs). Results: A significant decrease in iliac arterial blood flow, bladder wall blood flow, and bladder oxygen tension was found in the treated group. Bladder ischemia increased the frequency and amplitude of baseline spontaneous smooth muscle contractility. Ischemic tissues with urothelium (Uro+) demonstrated significant increases in the contractile response to electrical field stimulation (EFS) and carbachol relative to control Uro+ tissues. Urothelial removal increased smooth muscle contraction in the control tissues but had no significant effect in the ischemic/hypoxic tissues. Contraction of control tissues without urothelium (Uro-) was similar to contraction of ischemic Uro+ tissues. Contractions of ischemic Uro+ and control Uro- tissues were unchanged after treatment with the cyclooxygenase (COX) inhibitor indomethacin, while they were significantly reduced by the 5-lipoxygenase (5-LO) inhibitor NDGA. EIA showed no change in PGs release from the ischemic urothelium, but significant increase in PGF(2-alpha) and thromboxane A, release from the ischemic suburothelial tissue. Ischemia increased the release of LTB4, LTC4, and LTE4 from both urothelium and suburothelial tissue. Conclusions: Our studies Suggest loss of urothelial-mediated tone and LTs-mediated smooth muscle instability in the chronically ischemic/hypoxic bladder. Published 2004 Wiley-Liss, Inc(dagger).