TMEM16A (anoctamin 1, Ano1) is a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca²⁺-activated Cl^- channels. TMEM16A-null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 month after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca²⁺, but it is unclear whether Ca²⁺ binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, Ca²⁺ binding protein calmodulin appears indispensable and interacts physically with TMEM16A: Openers of small- and intermediate-conductance Ca²⁺, activated potassium channels know to interact with calmodulin such as 1-EBIO, DCEBIO, or riluzole, also activate TMEM16A. This reinforces the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.