In the 1970s, exposure to vinyl chloride (VC) was shown to cause liver angiosarcoma in VC workers. We have developed a new LC-MS/MS method for analyzing the promutagenic DNA adduct N(2),3-ethenoguanine (εG) and have applied this to DNA from tissues of both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days or 1100 ppm VC for 1 day. This assay utilizes neutral thermal hydrolysis and an HPLC cleanup prior to quantitation by LC-MS/MS. The number of endogenous and exogenous εG adducts in DNA from tissues of adult rats exposed to [(13)C(2)]-VC for 5 days was 4.1 ± 2.8 adducts/10(8) guanine of endogenous and 19.0 ± 4.9 adducts/10(8) guanine of exogenous εG in the liver, 8.4 ± 2.8 adducts/10(8) guanine of endogenous and 7.4 ± 0.5 adducts/10(8) guanine of exogenous εG in the lung, and 5.9 ± 3.3 adducts/10(8) guanine of endogenous and 5.7 ± 2.1 adducts/10(8) guanine of exogenous εG in the kidney (n = 4). Additionally, the data from weanling rats demonstrated higher numbers of exogenous εG, with ∼4-fold higher amounts in the liver DNA of weanlings (75.9 ± 17.9 adducts/10(8) guanine) in comparison to adult rats and ∼2-fold higher amounts in the lung (15.8 ± 3.6 adducts/10(8) guanine) and kidney (12.9 ± 0.4 adducts/10(8) guanine) (n = 8). The use of stable isotope labeled VC permitted accurate estimates of the half-life of εG for the first time by comparing [(13)C(2)]-εG in adult rats with identically exposed animals euthanized 2, 4, or 8 weeks later. The half-life of εG was found to be 150 days in the liver and lung and 75 days in the kidney, suggesting little or no active repair of this promutagenic adduct.