We evaluated the cytotoxic effect of conjugated linoleic acid (CLA) on rat hepatoma dRLh-84 cells in vitro. When cells were cultured in the presence of CLA, strong cytotoxic effect on dRLh-84 cells was recognized at 1 microM level compared to the control vehicle group, and trans10, cis12-CLA but not cis9, trans11-CLA was shown to be an active isomer for inducing this effect. Increase of the sub-G1 population and activation of caspase-3 and 9 accompanied with a time-dependent cleavage of poly(ADP-ribose) polymerase were recognized in dRLh-84 cells treated with trans10, cis12-CLA. In addition, we could see nuclear fragmentation in dRLh-84 cells treated with trans10, cis12-CLA by laser scanning confocal microscopy observation. Cytotoxic effect of trans10, cis12-CLA on normal hepatocytes was weaker than on dRLh-84 cells. These data indicate trans10, cis12-CLA has a potent cytotoxic effect on dRLh-84 cells through at least in part by an apoptotic pathway.