1 To examine the presence of nitric oxide synthase (NOS) activity in female dog urethra, pharmacological experiments were performed using electrical field stimulation (EFS), guanethidine, atropine, NG-nitro-L-arginine methyl ester and L-arginine, NOS immunohistochemistry using specific anti-NOS antibody, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining were also performed. 2 EFS caused frequency-dependent contractions in all urethral preparations, but in the presence of guanethidine and atropine, EFS caused significant relaxation in the proximal urethra and was without effect on the distal urethra. 3 In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, small contractions to EFS were re-established in the proximal urethra, but not in the distal urethra. NG-nitro-D-arginine methyl ester had no such effect. 4 In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, the addition of Larginine, restored the EFS-elicited relaxant responses previously seen with guanethidine and atropine alone in the proximal urethra (at 30 Hz; 12.89+5.27% to -2.44+4.43%, mean+s.e., P<0.05). DArginine had no such effect. 5 In the distal urethra, the addition of NG-nitro-L-arginine methyl ester and then L-arginine had no effect on responses to EFS in preparations treated with guanethidine and atropine. 6 Sodium nitroprusside caused relaxation in both the proximal and distal urethra. The relaxant responses per cm2 cross sectional area in the proximal and distal urethra were 1.23 + 0.29, and 2.02 + 0.54 g cm-2 cross sectional area (mean + s.e.), respectively: there was no significant difference between them. 7 Both NOS and NADPH diaphorase-positive neurones were present in dog urethra, the densities of both being higher in the proximal urethra than in the distal urethra. 8 These results show that female dog urethra possesses NOS nerves and that endogenous NO may play a role in relaxation in the proximal but not the distal urethra.