To clarify the relationship between DNA damage and free radical generation caused by smoking in vivo, DNA damage was investigated in the mouse lung by single-cell gel electrophoresis assay after exposure to cigarette smoke (CS) or gas phase cigarette smoke (GPCS). Although GPCS did not induce DNA lesions, bimodal peaks of DNA damage were detected in mouse lung exposed to CS, one immediately after exposure and another 15 min later. Pretreatment with a specific hydroxyl radical (•OH) scavenger completely prevented both types of DNA damage induced by CS. Electron spin resonance (ESR) study of the kinetics of free radical generation in CS or GPCS revealed that •OH could be detected immediately after the spin trapping of CS without chelators (first •OH generation), whereas •OH was also generated gradually with a time lag when the spin trapping was performed with chelators (second •OH generation). Our ESR study also indicated that the first •OH peak was probably generated from H(2)O(2) via a metal-independent pathway, whereas the second •OH peak might have been generated from H(2)O(2) and other sources via at least two different metal-masked pathways. The bimodal DNA damage induced in lung by smoking appears to be the result of a time lag between the first •OH generation and second •OH generation after exposure to the tar in CS.