Calcium-activated chloride channels (CaCCs) are involved in numerous physiological functions, including the epithelial movement of fluid. Anoctamin 1 (ANO1) has recently been cloned and characterized as a CaCC and is known to be expressed in various secretory epithelia and in nervous tissues such as the dorsal root ganglia and retina. However, data regarding the expression, function, and cellular and subcellular localization of CaCCs in the brain are still limited. We investigated the distribution and expression patterns of ANO1 in adult mouse brain. Reverse transcriptase plus the polymerase chain reaction, Western blot, and immunohistochemical analyses demonstrated that ANO1 was widely distributed throughout the brain. Furthermore, ANO1 was strongly expressed in two auditory brainstem nuclei: the medial nucleus of the trapezoid body (MNTB) and the anteroventral cochlear nucleus (AVCN). Double-labeling experiments revealed that this ANO1 expression was exclusive to the presynaptic endings of both the MNTB and AVCN. ANO1 is thus mainly localized at presynaptic terminals in various brain regions, specifically in two auditory brainstem nuclei, the MNTB and AVCN, and might therefore contribute to the high-frequency synaptic transmission of auditory signals.