Effects of bile acids on cystolic Ca++ activity and cell viability of isolated rat hepatocytes were studied to test the hypothesis that bile acids may produce hepatotoxicity by increasing cystolic Ca++ activity. Changes in cystolic Ca++ activity were calculated from time-dependent changes in fluorescence of quin-2 loaded hepatocytes. Release of lactate dehydrogenase and changes in propodium iodide fluorescence were used to assess cell viability. Bile acids studied were unconjugated and taurineconjugated cholate, chenodeoxycholate (and taurochenodeoxycholate), deoxycholate (and taurodeoxycholate) and lithocholate (and taurolithocholate). With the exception of cholate and taurocholate, bile acids increased cystolic Ca++ activity within 10 to 30 sec in a concentration-dependent fashion (0.05 to 1.0 mM) and in the order lithocholate = taurolithocholate > chenodeoxycholate = taurochenodeoxycholate = deoxycholate = taurodeoxycholate. The initial increase in cystolic Ca++ activity by bile acids was not due to cell damage, since bile acid-induced decreases in cell viability were not significant until 2 to 3 min. At higher concentrations of unconjugated bile acid, there was a secondary increase in quin-2 fluorescence corresponding temporally to the increase in propodium iodide fluorescence, indicating cell damage after the initial increase in cystolic Ca++ activity. The ability of conjugated and unconjugated bile acids to increase cystolic Ca++ activity was abolished and decreased (60 to go%), respectively, in the absence of extracellular Ca", indicating that extracellular Ca++ is the major source of the bile acid-induced increase in cystolic Ca++ activity. Conjugated bile acids at lower concentrations (0.2 m M taurolithocholate and 0.5 m M taurodeoxycholate and taurochenodeoxycholate), but not at higher concentrations (0.5 m M taurolithocholate and 1 .O m M taurodeoxycholate and taurochenodeoxycholate), failed to produce cell damage in the absence of extracellular Ca++. This study showed, for the first time, that bile acids increase cystolic Ca++ activity, and that the increase in cystolic Ca++ activity precedes cell damage. These results raise the possibility that the hepatotoxic effect of bile acids is, at least in part, related to their ability to increase cystolic Ca++ activity.