The role of extracellular Ca2+ ions in the killing of liver cells by CCl4 was studied in primary cultures of rat hepatocytes. The dependence of in vitro cell killing on the metabolism of CCl4 was first examined in order to document the similarity between the action of CCl4 on cultured hepatocytes and the action of CCl4 on liver cells in the intact animal. Cells prepared from male rats pretreated with phenobarbital were more sensitive to CCl4 than cells prepared from either male or female rats. The killing of hepatocytes by CCl4 was prevented by addition of SKF 525A to the culture medium. This protection was accompanied by evidence of decreased CCl4 metabolism as assessed by the extent of covalent binding of 14C-CCl4 metabolites to total cellular lipids and proteins, and by the extent of formation of conjugated dienes accompanying the peroxidation of phospholipids isolated from total cell lipids. The extent of killing of the hepatocytes by CCl4 was dependent on the Ca2+ concentration in the tissue culture medium. Total Ca2+ concentrations lower than 0.10 mM were not associated with any CCl4-induced cell death, and the number of dead cells increased with increasing Ca2+ from 0.3 to 3.6 mM. This dependency on extracellular Ca2+ was not due to dependency of the extent of metabolism of CCl4 on Ca2+. The Ca2+ concentration in the medium had no effect on the extent of covalent binding of metabolites of CCl4 to lipids and to proteins and on the extent of peroxidation of phospholipids as shown by the formation of conjugated dienes. In addition, hepatocytes incubated in low Ca2+ with CCl4 developed further evidence of cell injury, as indicated by the killing of these cells following the addition of high Ca2+ concentrations under conditions prohibiting any further metabolism of the CCl4. The results of this study indicate that it is the presence of extracellular Ca2+ that converts initially nonlethal cell injury into irreversible cell injury in CCl4-treated cells. This action of Ca2+ most likely represents an influx into the cell across an injured permeability barrier at the plasma membrane, in accord with the accumulation of large quantities of Ca2+ in CCl4-intoxicated liver cells in the intact animal. The relation between this alteration in Ca2+ homeostasis and the metabolism of CCl4 is discussed.