The nitric oxide (NO) synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA), but not D-NMMA, inhibited the NANC-mediated relaxations of the rat anococcygeus muscle, but did not affect the relaxation produced by sodium nitroprusside. 2. The inhibitory effect of L-NMMA was reversed by L-arginine but not by D-arginine, and prior exposure to L-arginine blocked the effect of L-NMMA. 3. The noradrenergically mediated contractions of the anococcygeus elicited by field stimulation were slightly enhanced by L-NMMA, but the response to noradrenaline was not affected. 4. The results suggest that NANC transmission in the rat anococcygeus muscle involves the generation of NO from arginine.