Activation of an apical Ca²⁺-activated Cl⁻ channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl⁻ current and Cl⁻ efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl⁻ channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A^−/−). Ca²⁺-dependent salivation was abolished in Tmem16A^−/− mice, demonstrating that Tmem16A is obligatory for Ca²⁺-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A^−/− mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr^{∆F508/∆F508}) or ClC-2 (Clcn2^−/−) Cl⁻ channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl⁻ channel. Indeed, Cl⁻ channel blockers abolished fluid secretion, indicating that Cl⁻ channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic–induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A^−/− mice identify Tmem16A as the Cl⁻ channel essential for muscarinic, Ca²⁺-dependent fluid secretion in adult mouse salivary glands.