Objective: To investigate the properties of the smooth muscle layers in the urethral wall of male and female greyhounds, and to consider their roles in continence and micturition. Materials and methods: The distribution and innervation of the smooth muscle layers of the prostate capsule and membranous urethra of male greyhounds were assessed. Strips of smooth muscle from these regions were used to determine the neuropharmacological properties by assessing the excitatory and inhibitory responses to nerve stimulation, and the effects of blocking agents. These were compared with strips from the proximal urethra and from the female urethra. Results: The smooth muscle of the membranous urethra comprised 9% of the wall and received its innervation exclusively in branches from the pelvic plexus. The cholinergic innervation in the male produced 80% of the total contractile response in the longitudinal membranous urethra, 50% in the prostate capsule and 13% in the circular muscle of the proximal urethra. In the female all areas had poor contractile responses. Inhibitory fibres produced relaxation in all parts of male and female urethrae with the major effect caused by nitric oxide. Adrenergic nerves contributed to both residual excitation (α receptors) and inhibition (β receptors). Conclusions: The longitudinal smooth muscle of the male membranous urethra probably shortens the urethra during micturition, through the activity of cholinergic nerves, whereas the circular smooth muscle of the proximal urethra, under adrenergic control, may be contracted during continence and ejaculation. In the female, the smooth muscle plays a minor role.