The immune cells use reactive oxygen species (ROS) for carrying out their normal functions while an excess amount of ROS can attack cellular components that lead to cell damage. In the present study, peritoneal macrophages (6 x 10(6) cells, >95% viable) isolated from male Swiss mice were treated with nicotine (1 mM, 5 mM, 10 mM, 25 mM, and 50 mM) in vitro for 12 h and the superoxide anion generation, lipid peroxidation, protein oxidation and antioxidant enzymes status were monitored. Maximum superoxide radical generation was found at the dose of 10 mM nicotine. The lipid peroxidation and protein oxidation were increased significantly (p < 0.05) along with the increasing dose of nicotine. The reduced glutathione level, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase activities were decreased significantly (p < 0.05), and oxidized glutathione level was increased significantly (p < 0.05) with the increasing dose of the nicotine. From these experiments, it was also observed that all the changes in peritoneal macrophages with 10 mM, 25 mM, and 50 mM nicotine had no significant difference. To observe the effect of nicotine in vivo, this study examined the liver and spleen antioxidant status after nicotine administration (1 mg/kg BW) intraperitoneally in mice and found the diminished SOD activity and GSH level. It may be concluded that nicotine is able to enhance the production of ROS that produced oxidative stress in murine peritoneal macrophages. It also suggested that, 10 mM in vitro nicotine treatment for 12 h is the effective dose.