This study was performed to evaluate the isomer-specific cytotoxic effects of conjugated linoleic acid (CLA) on rat hepatoma dRLh-84 cells in vitro. A 10trans,12cis (10t,12c)-CLA showed a strong cytotoxic effect on dRLh-84 cells in culture, whereas no such effect was observed with 9cis,11trans (9c,11t)-CLA or linoleic acid. The optimum concentration for induction of cytotoxity by 10t,12c-CLA was 5 to 10 μM, but the effect was alleviated at higher concentrations. Coincubation with oleic or palmitoleic acid and 10t,12c-CLA cancelled the cytotoxic effect, but other major saturated or polyunsaturated fatty acids and eraidic acid did not interfere with 10t,12c-CLA–induced cytotoxity. The cytotoxic effect was also alleviated by α-tocopherol (α-toc) and α-tocotrienol but not by any other antioxidant regent examined. Significant cytotoxity of 10t,12c-CLA was detected after only a 15-min incubation, and the most noticeable effect was seen after 3 h. After incubation with 10t,12c-CLA at 10 μM, an additional 90 μM of 10t,12c-CLA or 100 μM of α-toc was also able to alleviate the cytotoxity. When cells were treated with 10 μM 10t,12c-CLA for more than 48 h, treatment with additional CLA or α-toc could not prevent cell death.