1. The effects of two inhibitors of nitric oxide synthase, NG-monomethyl L-arginine (L-NMMA) and NG-nitro L-arginine (L-NOARG), were examined on non-adrenergic non-cholinergic (NANC) inhibitory transmission in the rat anococcygeus, bovine retractor penis (BRP) and bovine penile artery. 2. In the rat anococcygeus, L-NMMA (10-1000 microM) produced a concentration-dependent augmentation of guanethidine (30 microM)-induced tone and inhibited NANC relaxation at all frequencies tested (0.1-20 Hz): the maximum inhibition obtained was 56 +/- 6% (n = 6). L-NOARG (0.3-30 microM) also augmented tone and inhibited NANC relaxation in a concentration-dependent manner, but unlike L-NMMA the maximum inhibition was 100%. 3. In the BRP, L-NMMA (10-100 microM) had no effect on tone or NANC-induced relaxation, but at 1000 microM tone was increased and NANC relaxation inhibited by 25 +/- 7% (n = 6). L-NOARG (0.3-30 microM) produced a concentration-dependent increase in tone and inhibition of NANC relaxation. As in the rat anococcygeus, inhibition of NANC relaxation was complete. 4. The effects of L-NMMA and L-NOARG were stereospecific since D-NMMA (10-1000 microM) and D-NOARG (1-1000 microM) had no effect on tone or NANC relaxation of the rat anococcygeus or BRP. 5. L-Arginine (10-300 microM) had no effect by itself on NANC-induced relaxation of the rat anococcygeus or BRP. It did, however, reverse the ability of L-NMMA (10-1000 microM) to augment tone and inhibit NANC relaxation in the rat anococcygeus and BRP. 6. On the bovine penile artery, both L-NMMA (100 microM) and L-NOARG (30 microM) augmented the tone induced by guanethidine (30 microM) and 5-hydroxytryptamine (0.2 microM) in an endothelium-dependent manner. L-NMMA had no effect on NANC-induced relaxation, but inhibited acetylcholine-induced endotheliumdependent relaxation. L-NOARG abolished NANC relaxation at all frequencies tested and inhibited acetylcholine-induced relaxation. D-NOARG (30 microM) had no effect on NANC or acetylcholine-induced relaxation. 7. The ability of L-NOARG to abolish NANC-induced relaxation in the rat anococcygeus, BRP and bovine penile artery suggests that the L-arginine-nitric oxide pathway mediates neurotransmission in all three tissues. The effectiveness of L-NMMA in blocking NANC relaxation-in the rat anococcygeus but not the BRP and bovine penile artery suggests a species difference in the neuronal nitric oxide synthase. The neuronal and endothelial nitric oxide synthases in the penile artery also appear to differ.