Exposure of cultured hepatoma 1c1c7 cells to KCN and iodoacetate, to produce chemical anoxia, caused a rapid and sustained increase in cytosolic‐free Ca2+concentration, which was associated with depletion of intracellular ATP and glutathione. These changes occurred before the loss of cell viability and were accompanied by the appearance of plasma membrane blebs. Pretreatment of the cells with the Ca2+chelators Quin 2 or BAPTA markedly delayed both the onset of blebbing and loss of cell viability, but did not affect KCN‐ and iodoacetate‐induced loss of ATP and glutathione. Together, these results strongly suggest that a sustained increase in cytosolic Ca2+concentration plays an important role in killing of hepatoma cells by chemical anoxia.— Nicotera, P.; Thor, H.; Orrenius, S. Cytosolic‐free Ca2+and cell killing in hepatoma 1c1c7 cells exposed to chemical anoxia.FASEB J.3: 59‐64; 1989.