The release of bladder-derived relaxant factor in a coaxial bioassay system and the effects of reactive oxygen species were studied. After precontraction with phenylephrine (10−6–3×10−6) or 50 mM K+, acetylcholine (10−8–10−3 M) induced relaxation in rat anococcygeus muscle mounted within rat bladder in a tissue bath. This relaxation was not altered by the removal of the urothelium or incubation with tetrodotoxin (10−6 M). However, bupivacaine (10−4 M) and lidocaine (3×10−4 M) inhibited this response after raising the pH of the nutrient solution to 7.8, and oxybuprocaine (10−4 M) exerted inhibitory effect at both physiological pH (7.4) and at pH 7.8. Exposure to electrolysis-generated reactive oxygen species or incubation with hydrogen peroxide and pyrogallol did not alter the acetylcholine response. Present results indicate that the bladder-derived relaxant factor does not behave like endothelium-derived hyperpolarizing factor, but its release may be associated with tetrodotoxin-resistant Na+ channels, which are probably in the neurons of the bladder rather than in the urothelium or detrusor muscle. Furthermore, reactive oxygen species do not interact with this relaxing factor, the exact nature and the physiological importance of which, however, remains to be established.