Ano1 is a recently discovered Ca(2+)-activated Cl(-) channel expressed on interstitial cells of Cajal (ICC) that has been implicated in slow-wave activity in the gut. However, Ano1 is expressed on all classes of ICC, even those that do not contribute to generation of the slow wave, suggesting that Ano1 may have an alternate function in these cells. Ano1 is also highly expressed in gastrointestinal stromal tumors. Mice lacking Ano1 had fewer proliferating ICC in whole mount preparations and in culture, raising the possibility that Ano1 is involved in proliferation. Cl(-) channel blockers decreased proliferation in cells expressing Ano1, including primary cultures of ICC and in the pancreatic cancer-derived cell line, CFPAC-1. Cl(-) channel blockers had a reduced effect on Ano1(-/-) cultures, confirming that the blockers are acting on Ano1. Ki67 immunoreactivity, 5-ethynyl-2'-deoxyuridine incorporation, and cell-cycle analysis of cells grown in low-Cl(-) media showed fewer proliferating cells than in cultures grown in regular medium. We confirmed that mice lacking Ano1 had less phosphorylated retinoblastoma protein compared with controls. These data led us to conclude that Ano1 regulates proliferation at the G(1)/S transition of the cell cycle and may play a role in tumorigenesis.