The effect of the competitive inhibitor of L-arginine, NG-monomethyl L-arginine (L-NMMA) on the response of the rat anococcygeus muscle to non-adrenergic, non-cholinergic (NANC) inhibitory nerve stimulation has been examined. L-NMMA causes a rise in muscle tone and inhibition of the response to nerve stimulation. The stereoisomer D-NMMA is without effect. The rise in tone and inhibition of the nerve response is reversed by L-arginine. Another analogue, L-canavanine, which is effective against L-arginine utilization in the macrophage, was without effect on the rat anococcygeus. These results provide indirect evidence for nitric oxide (NO) or a substance releasing NO as the transmitter of the NANC nerves in this tissue.