Cigarette smoking is associated with the development of numerous human cancers in various tissues. Cigarette smoke is a complex mixture of chemicals and contains more than 50 known or probable human carcinogens. The majority of carcinogens present in cigarette smoke require metabolism to form the reactive species that have the potential to react with DNA, resulting in the formation of DNA adducts. Interestingly, it has been recently suggested that there was an uncharacterized direct-acting ethylating agent present in cigarette smoke [1]. Since alkylation at the N-7 position of guanine in DNA is the predominant reaction site, N7-alkylguanine represents a good biomarker for determining exposure to alkylating agents. In our previous study, N7-ethylguanine (N7-EtG) has been detected in human urine and was found to be highly associated with cigarette intake of smokers [2]. But the source of this direct-acting ethylating agent in tobacco smoke is still unclear. In the present study, we aimed to investigate the characteristics of the direct-acting ethylating agent present in cigarette smoke by measuring the formation of N7-EtG following exposure of DNA to cigarette smoke in vitro. Cigarettes were smoked as specified under the Federal Trade Commission (FTC) standard conditions. Both the particulate and vapor phases of mainstream and sidestream smoke were collected. After sampling, the aqueous extracts of cigarette smoke were added to calf thymus DNA and the mixtures were incubated. The levels of N7-EtG formed were then quantitated by liquid chromatography tandem-mass spectrometry (LC-MS/MS) with on-line solid phase extraction [3]. Our results showed that N7-EtG was formed in vitro and was highly correlated with the number of cigarettes treated. Moreover, this uncharacterized direct-acting ethylating agent was found to be mainly present in the particulate phase of mainstream smoke, indicating it poses a greater health threat to smoker themselves than others.