Anion transport by the colonic mucosa maintains the hydration and pH of the colonic lumen, and its disruption causes a variety of diarrheal diseases. Cholinergic agonists raise cytosolic Ca2+ levels and stimulate anion secretion, but the mechanisms underlying this effect remain unclear. Cholinergic stimulation of anion secretion may occur via activation of Ca2+-activated Cl- channels (CaCCs) or an increase in the Cl- driving force through CFTR after activation of Ca2+-dependent K+ channels. Here we investigated the role of a candidate CaCC protein, bestrophin-2 (Best2), using Best2-/- mice. Cholinergic stimulation of anion current was greatly reduced in Best2-/- mice, consistent with our proposed role for Best2 as a CaCC. However, immunostaining revealed Best2 localized to the basolateral membrane of mucin-secreting colonic goblet cells, not the apical membrane of Cl--secreting enterocytes. In addition, in the absence of HCO3-, cholinergic-activated current was identical in control and Best2-/- tissue preparations, which suggests that most of the Best2 current was carried by HCO3-. These data delineate an alternative model of cholinergic regulation of colonic anion secretion in which goblet cells play a critical role in HCO3- homeostasis. We therefore propose that Best2 is a HCO3- channel that works in concert with a Cl:HCO3- exchanger in the apical membrane to affect transcellular HCO3- transport. Furthermore, previous models implicating CFTR in cholinergic Cl- secretion may be explained by substantial downregulation of Best2 in Cftr-/- mice.