Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST, which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in mouse lung and aorta. Exposure to tobacco smoke for 3 days (5h/day) decreased total plasma protein. These changes were exaggerated in GSTP^-/- mice. Aortic rings isolated from tobacco smoke-exposed GSTP^-/- mice showed greater attenuation of acetylcholine-evoked relaxation than those from GSTP^+/+ mice. Lung, plasma and aorta of mice exposed to tobacco smoke or acrolein (for 5h) accumulated more acrolein-adducted proteins than these tissues of mice exposed to air, indicating that exposure to tobacco smoke results in systemic delivery of acrolein. Relative to GSTP^+/+ mice, modification of some proteins by acrolein was increased in the aorta of GSTP^-/- mice. Aortic rings prepared from GSTP^-/- mice inhaling acrolein (1 ppm, 5h/day for 3 days) or those exposed to acrolein in the organ bath showed diminished acetylcholine-induced arterial relaxation more strongly than GSTP^+/+ mice. Acrolein-induced endothelial dysfunction was prevented by pretreating the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.