Unbumt tobacco and tobacco smoke contain a variety of carcinogens, exposure to which are causally associated with the incidence of several human cancers. Herein, we used isotope-dilution LC-MS/MS for the quantification of alkylated purines in DNA, following in vitro exposure to aqueous extracts of tobacco itself, and tobacco smoke. Our results demonstrated the presence of direct-acting ethylating agent(s) in unburnt tobacco, which 4.0-6.3 times exceeded that in the particulate phase of sidestream cigarette smoke and 6.8-8.9 times exceeded that in mainstream smoke. Interestingly, particulate phase of sidestream cigarette smoke exhibited higher ethylating potency than that in mainstream smoke. This finding refutes the previous assumptions that the ethylating agent (s) associated with smoking, are derived from cigarette smoke. Indeed, our data show that combustion of tobacco actually decreases the ethylating potency of tobacco. Although the identity of this agent(s) remains unknown, our data suggest that it is highly hydrophilic, and hence likely to be easily extracted by saliva. This would allow intimate contact with the tissues of the oropharyngeal cavity. Taken together, these results have profound implications for tobacco use, in particular for tobacco chewers and passive smokers, whose exposure to ethylating agent(s) is greater than previously thought.