In this study, we examined the possibility that 5-HT_1A receptors may underlie sexually dimorphic mechanisms affecting regulation of urethral functions in anesthetized rats. Simultaneous recordings of the intravesical pressure under isovolumetric conditions (isovolumetric IVP), external urethral sphincter-electromyography (EUS-EMG), and urethral perfusion pressure (UPP) were used to examine the effects of a 5-HT_1A receptor agonist (8-OH-DPAT) and antagonist (WAY-100635) on bladder and urethral functions. This research also evaluated the effects of 8-OH-DPAT and α-bungarotoxin (a neuromuscular blockade agent) on urethral continence using leak point pressure (LPP) testing, and the distribution of 5-HT_1A receptors in the lower urinary tract was assessed by immunohistochemistry (IHC). The serotonergic mechanism that controls the urinary bladder and EUS-EMG activity showed no significant sexual difference, but urethral activity in UPP and LPP values exhibited some sexual differences. 8-OH-DPAT enhanced urethral pressure during continence in rats of both sexes, but the drug elevated the pressure during voiding in males and reduced it in females. The distribution of 5-HT_1A receptors in the spinal cord also showed some sexual differences. The present study contributes to our understanding of the role of 5-HT_1A receptors in physiological and IHC properties of urethral smooth muscle in rats of different sexes. These findings may be a basis for the future development of pharmacotherapies for stress urinary incontinence in men.