Our previous studies demonstrated that manumycin A, a farnesyltransferase inhibitor, induced apoptosis of anaplastic thyroid cancer cells via the intrinsic apoptosis pathway and induced reactive oxygen species (ROS), which mediated DNA damage. In this study, we investigated the hypothesis that the mechanism of apoptosis induced by manumycin in anaplastic thyroid cancer cells fits the general pattern of the "xenobiotic apoptosis pathway," the hallmarks of which are induction of oxidative stress, mitogen-activated protein kinase (MAPK) signaling, and cytochrome c release, which activates the intrinsic apoptosis pathway. We found that manumycin reduced intracellular glutathione and generated ROS: nitric oxide and superoxide anions. Manumycin-induced apoptosis correlated with increase in ROS. Quenching of ROS with N-acetyl-L-cysteine prevented cytochrome c release by manumycin. Manumycin induced phosphorylation of p38 MAPK, which was blocked by N-acetyl-L-cysteine. p38 MAPK may be an important signaling mediator in the activation of the intrinsic apoptotic pathway by manumycin because the p38 MAPK inhibitor SB203580 inhibited cytochrome c release and activation of caspase-3 by manumycin. In conclusion, manumycin activated the intrinsic apoptosis pathway via activation of p38 MAPK by oxidative stress. The mechanism of apoptosis induced by manumycin fits the emerging general pattern for apoptosis induced by xenobiotics.