Soluble and particulate fractions from rabbit urethra converted [14C]arginine to [14C]citrulline, indicating the presence of nitric oxide synthase activity in these fractions. Both soluble and particulate nitric oxide synthase activities were NADPH dependent, and the soluble activity was Ca2+ dependent. Three nitric oxide synthase (NOS) inhibitors affected transmural nerve stimulation induced relaxation responses in the rabbit urethra and the activity of soluble nitric oxide synthase with the same rank order of potency, i.e., NG-nitro-L-arginine (NNA) > NG-methyl-L-arginine (NMA) > canavanine (CAN). The rank order of potency with respect to particulate NOS activity was CAN > NMA = NNA. The relaxation responses to electrical stimulation were accompanied by increases in cyclic GMP. These results suggest that NOS activity found in the soluble fraction of urethral homogenates produces nitric oxide that in turn increases cyclic GMP levels which mediates the relaxation responses induced by transmural nerve stimulation in the rabbit urethra.