2,5‐Hexanedione (2,5‐HD) pretreatment potentiated CHCl3‐induced hepatotoxicity. 2,5‐HD significantly increased hepatic cytochrome P‐450, NADPH cytochrome c reductase, aniline hydroxylation, p‐nitroanisole O‐demethylation, and aminopyrine N‐demethylation in both male and female mice. 2,5‐HD pretreatment potentiated CHCl3‐induced centrilobular necrosis and increased serum alanine amino transferase (ALT) activity by 20 times more than CHCl3alone. Similarly, 2,5‐HD pretreatment potentiated CDCl3‐induced hepatotoxicity as well as CCl4‐induced hepatotoxicity in male mice, but did not potentiate trichloroethylene‐, 1,1,2‐trichloroethane‐, or perchloroethylene‐induced hepatotoxicity. In female mice, 2,5‐HD pretreatment potentiated CHCl3‐ and CDCl3‐induced hepatotoxicity as well as trichloroethylene‐, 1,1,2‐trichloroethane‐, and carbon tetrachloride‐induced hepatotoxicity, but not perchloroethylene‐induced hepatotoxicity. 2,5‐HD pretreatment had no preferential effect on either CHCl3‐ or CDCl3‐induced hepatotoxicity in females. However, phenobarbital pretreatment did differentiate CHCl3‐ and CDCl3‐induced hepatotoxicity in females. 2,5‐HD‐induced potentiation of halocarbon hepatotoxicity is sex dependent.