1. Mechanical recordings were made in vitro from circularly oriented strips of the bladder neck muscle of sheep. In the absence of drugs, electrical field stimulation at frequencies of 0.2-1 Hz evoked clear-cut relaxations throughout 1 min stimulation periods, while higher stimulus frequencies (2-8 Hz) evoked variable responses consisting of relaxation, contraction or a mixture of both. All of the responses were abolished by tetrodotoxin (10(-6) M). 2. The contractions were reduced by guanethidine (10(-6) M) and atropine (10(-6) M), so that in the presence of these drugs clear-cut relaxations were obtained at 0.2-8 Hz stimulation, indicating that the relaxations were mediated by non-adrenergic, non-cholinergic (NANC) nerves. 3. The NANC relaxations were blocked by L-NG nitro arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis. The antagonism by L-NAME was reversed by L-arginine. 4. Another feature of the NANC relaxation was 'rebound contraction' which occurred when the stimulus was switched off. The rebound contraction was also blocked by L-NAME and restored by L-arginine. 5. The relaxations and rebound contractions were unaffected by either alpha,beta-methylene ATP (10(-5) M) or 2-methylthio ATP (10(-5) M). 6. S-Nitroso-L-cysteine, a substance which spontaneously releases NO at physiological pH, mimicked the relaxation and rebound contraction produced by nerve stimulation. 7. It is concluded that nerve-evoked relaxation of the bladder neck is mediated by NO, or a closely related substance such as S-nitroso-L-cysteine.