The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Use of chewing tobacco is extremely popular in Far East and Middle East countries. In some of these countries, these chewing tobaccos are mixed with areca nut, lime and catechu and sold as pan masala. In this study, we examined three different forms of commercially available pan masala, and examined the development of cytotoxicity using primary culture of normal human oral keratinocytes (NHOKs). NHOK cells were treated with three different forms of pan masala, gutkha, pan masala with saccharin and pan masala as well as Kentucky smokeless chewing tobacco (STE) and the development of oxidative stress, and DNA damage were monitored. The results of our study demonstrated significant amount of the superoxide anion production, lipid peroxidation, DNA fragmentation and DNA ladders with all of the chewing tobacco materials tested. Laser scanning microscopy revealed concentration-dependent effects of STE on the modulation of intracellular redox states. The results, thus, document that the cytotoxic effects of the chewing tobaccos including pan masalas are mediated through the production of the reactive oxygen species.