1. The effects and interactions of a series of NG-substituted analogues of L-arginine known to inhibit nitric oxide synthase were examined on non-adrenergic, non-cholinergic (NANC) neurotransmission in the bovine retractor penis (BRP) and rat anococcygeus muscles. 2. Treatment of BRP muscle strips with either NG-nitro L-arginine (L-NOARG: 0.1-10 microM) or NG-nitro L-arginine methyl ester (L-NAME; 0.1-100 microM) produced a concentration-dependent blockade of NANC relaxation: blockade was complete at the highest concentration of each. 3. Pretreatment with L-arginine (1-10 mM) had no effect on NANC relaxation by itself, but inhibited, in a concentration-dependent manner, the subsequent ability of both L-NOARG (0.1-300 microM) and L-NAME (0.1-1 mM) to produce blockade. L-Arginine (1-10 mM) reversed established submaximal blockade of NANC relaxation induced by L-NOARG (1 microM) or L-NAME (1 microM), but had little effect on maximal blockade induced by these agents. 4. In contrast to L-NOARG and L-NAME, NG-monomethyl L-arginine (L-NMMA; 1 microM-1 mM) had no effect by itself on NANC relaxation of the BRP. L-NMMA (0.1-1 mM) did, however, like L-arginine, inhibit, in a concentration-dependent manner, the subsequent ability of both L-NOARG (0.1-1 mM) and L-NAME (0.1-3 mM) to produce blockade, but was more potent. As with L-arginine, L-NMMA (0.1-1 mM) reversed established submaximal blockade of NANC relaxation induced by L-NOARG (1 microM) or L-NAME (1 microM), but had little effect on maximal blockade induced by these agents. 7. These findings suggest a complex series of interactions between L-arginine and certain of its NG-substituted analogues that are commonly used to inhibit nitric oxide synthase. The most striking new finding is that L-NMMA does not block NANC relaxation in the BRP, but acts with greater potency than the endogenous substrate, L-arginine, to inhibit the blockade induced by L-NOARG or L-NAME.Even on rat anococcygeus where L-NMMA acts as a partial blocker of NANC relaxation, further blockade by L-NOARG is inhibited.