The effect of urinary bladder inflammation on the activity of a bladder-derived relaxant factor in the coaxial bioassay system was examined. Bladder inflammation was induced by intraperitoneal (i.p.) cyclophosphamide or intravesical lipopolysaccharide (LPS) injection to male rats. In precontracted rat anococcygeus muscle that was placed within rat bladder (coaxial bioassay system), acetylcholine induced a relaxation response, which was not altered by the denudation of urothelium or incubation with indomethacin and N-G-methyl-L-arginine. Acetylcholine-induced relaxation was significantly attenuated, when bladders were removed from cyclophosphamide- and LPS-pretreated group of rats and were used with intact urothelium in the coaxial bioassay system. However, the impairment acetylcholine response in both pretreatment groups was not observed after denudation of the bladder urothelium. These results showed that bladder inflammation did not alter the synthesis and/or release of this bladder-derived relaxant factor, which is neither a cyclooxygenase product nor nitric oxide, but restricted its demonstration by coaxial bioassay assembly probably due to inflammation-induced mucosal oedema.