Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-induced AEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (ΔΨ_m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces AR after a 4 h exposure period. Further, the AT after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell ΔΨ_mand attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell ΔΨ_mafter 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos. (Mol Cell Biochem 234/235: 153–160, 2002)