The role of Ca2+ in the initiation of carbon tetrachloride (CCl4) hepatotoxicity was studied using perfused livers isolated from phenobarbital-pretreated rats in a single-pass system. Krebs-Henseleit bicarbonate buffer containing 1.3 mM CaCl2 (KHB) was the regular ionic milieu. In the liver perfused with fructose-supplemented regular KHB equilibrated with 95% N2-5% CO2, infusion of 0.5 mM CCl4 caused an early uptake of Ca2+ coupled with K+ leakage and Na+ uptake within the infusion time of 30 min, which was followed by a marked lactic dehydrogenase (LDH) leakage into the effluent perfusate and further Ca2+ uptake by the liver. With Ca(2+)-free medium, the prenecrotic K+ leakage and the successive LDH leakage were suppressed markedly. However, a perfusate exchange from regular to Ca(2+)-free KHB at the end of the prenecrotic stage did not protect against the LDH leakage, and the perfusate exchange conversely did not produce LDH leakage. Perfusion of the liver with high K+(Cl-) medium under 20% O2 markedly suppressed CCl4-induced LDH leakage even in the presence of Ca2+, whereas once CCl4 had acted under regular KHB perfusion, changing the medium to high K+ did not further prevent the LDH leakage. High K(+)-lactobionic acid medium containing Ca2+ and supplemented with fructose also suppressed LDH leakage under 95% N2 without the accompanying prenecrotic Ca2+ uptake. However, a change of the medium after CCl4 infusion to regular KHB containing Ca2+ caused LDH leakage and K+ leakage, with Ca2+ uptake. The prevention of LDH leakage in a different ionic milieu may not be due to suppression of CCl4 bioactivation, since the liver cytochrome P450 content decreased to a similar extent. These findings suggest that entry of extracellular Ca2+ into hepatocytes coupled with K+ leakage and Na+ entry is a prerequisite for CCl4-induced hepatocyte death and that association of Ca2+ with a CCl4-derived radical-mediated process may be necessary for early and irreversible plasma membrane damage.