Purpose: The predominant β-adrenoceptor subtype present in the bladder and urethra is β3-adrenoceptors. We investigated the role of β-adrenoceptors in mediating relaxation of the in vitro female pig urethra. Materials and Methods: Circular strips of urethral tissues were pre-contracted with KCl. Concentration-relaxation curves (CRCs) to β-adrenoceptor agonists were obtained in the absence and presence of antagonists. Results: The nonselective β-agonist isoproterenol in 30 animals and the β3-adrenoceptor agonist BRL37344 in 4 relaxed with high potency (pEC50 7.2 and 8.1, respectively), while the β2-adrenoceptor agonist salbutamol in 6 had low potency (pEC50 6.1). Mean maximal relaxation responses of BRL37344 and salbutamol relative to maximal isoproterenol responses were 89.8% and 76.7%, respectively. Propranolol (10 to 100 nM) in 18 animals antagonized CRCs to isoproterenol with high affinity (apparent pKB 8.6) but the Schild plot had a slope that was significantly less than unity (0.68, p <0.01). High concentrations of the β1-antagonist CGP20712A (3 to 30 μM) in 12 animals had no effect on responses to isoproterenol. The β2-antagonist ICI118551 (30 to 300 nM) in 25 animals antagonized responses to isoproterenol with high affinity (apparent pK B 8.03) with a Schild slope not different from unity (0.79). The β3-antagonist SR59230A (10 to 100 nM) in 12 animals antagonized CRCs to isoproterenol with an apparent pKB of 7 and with a Schild slope that was again significantly less than unity (0.62, p <0.01), indicating that responses to isoproterenol are mediated by more than 1 β-adrenoceptor subtype. According to the Schild plot of unity ICI118551 (3 to 30 nM) in 18 animals competitively antagonized responses to salbutamol with high affinity (pA2 8.5). Conclusions: In the pig urethra β-adrenoceptor mediated relaxations to isoproterenol are mediated via β2 and β3-adrenoceptors, while responses to β 2-adrenoceptor agonists such as salbutamol appear to be mediated only via β2-adrenoceptors.