Mitotane [1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane], an antineoplastic agent for inoperable adrenal carcinoma, was studied for its cytotoxic activity on a clonal line of feminizing human adrenal neoplastic cells (Fang-8 cells) in culture. At concentrations higher than 1.68 × 10-4 m, mitotane produced a dose-related toxic effect on the cells. The effect of the drug was specific to Fang-8 cells because the same treatment produced little or no toxicity on lines of rat pituitary GH3 cells and human skin fibrocytes. The effect of mitotane to Fang-8 cells was a reversible one. Electron microscopic pictures revealed that the drug was causing mitochondrial degeneration. In this testing system, several isomers and analogs of mitotane were found equally or more toxic than was mitotane itself. The dichloro- or trichloroethylene structure was essential for the cytotoxic activity while the chloro substituents on phenyl rings appeared to be unimportant. This system appears to be useful in elucidating the biochemical mechanism of mitotane action on adrenal cancer.