The effects of osmotic stress onchloride(CI-)currentsin the humanneuroblastomacell line CHP-100 were evaluated. Following exposure to hypoosmotic solution, an increase in whole-cell CI- current was observed. This current was blocked by the CI- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). Incellsloaded with the CI- permeability marker 125I, exposure to hypoosmotic solution increased 125I efflux by 197 +/- 14% (n = 41, p < 0.05) over controls. This increase was sensitive to NPPB. Hypoosmotic stress also increased cytosolic calcium levels (Ca2+) in fura-2-loadedcells. Pretreatment with EGTA inhibited the increase in cytosolic Ca2+, 125I efflux, and whole-cell CI- current produced by hypoosmotic solution. Antagonists of N-, L-, and T-type Ca2+ channels did not alter stimulation in 125I efflux or cytosolic Ca2+ levels during osmotic stress. However, omega-conotoxin MVIIC, a P-type Ca2+ channel blocker, inhibited hypoosmotically activated whole-cell CI-currentsand increases in cytosolic Ca2+. It is concluded that a Ca(2+)-dependent change in CI- permeability is activated in CHP-100cellsin response to osmotic stress.