<p>Anoctamin-1 Ca²⁺-activated Cl⁻ channels (ANO1) are proposed to modulate contractility of urethra smooth muscle cells (USMC), but their cellular expression and contribution to agonist/neural evoked activity is unclear. ANO1 is implicated as a potential target to treat incontinence, thus this is an important issue to resolve. We sought to clarify roles of ANO1 in contractility of mouse USMC. We found expression of <i>Ano1</i> transcripts in murine urethra, with no difference between male and females. Immunolabelling revealed ANO1 was expressed in USMC and not in specialized populations of interstitial cells (c-kit⁺ interstitial of Cajal-like cells (ICC-LC) and PDGFRα⁺ cells). However, a specific ANO1 channel inhibitor, Ani9, failed to affect urethral contractions elicited by phenylephrine, arginine vasopressin or electrical field stimulation of intrinsic nerves. CaCC_inhA01 also failed to affect urethral contractions. In addition, Ani9 had no effect on Ca²⁺ signals generated by USMC in situ. In contrast, Ani9 effectively reduced spontaneous contractions and Ca²⁺ signals of mouse proximal colon. In addition, Ani9 inhibited ANO1 currents recorded in HEK 293 cells, at concentrations 30 times less than those used in organ bath experiments. Our data suggest that despite expression of ANO1 in USMC, these channels do not contribute to basal Ca²⁺ signalling, or agonist and neurally-evoked contractions in murine urethra.</p>