Chenodeoxycholate is toxic to hepatocytes, and accumulation of chenodeoxycholate in the liver during cholestasis may potentiate hepatocellular injury. However, the mechanism of hepatocellular injury by chenodeoxycholate remains obscure. Our aim was to determine the mechanism of cytotoxicity by chenode- (Ca,2.) was observed. Removal of extracellular Ca2" abolished the rise in Ca,2., decreased cellular proteolysis, and protected against cell killing by glycochenodeoxycholate. The results suggest that glycochenodeoxycholate cytotoxicity results from ATP depletion followed by a subsequent rise in Ca,". The rise in Ca,2. leads to an increase in calcium-dependent degradative proteolysis and, ultimately, cell death. We conclude that glycochenodeoxycholate causes a bioenergetic form of lethal cell injury dependent on ATP depletion analogous to the lethal cell injury of anoxia. (J. Clin. Invest. 1993. 92:17-24.)