Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) in DNA to generate a pair of diastereomeric 1,N2- propanodeoxyguanosine adducts (Cro-dGuo, 2), which occur in (6S,8S) and (6R,8R) configurations. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Cro-dGuo adducts inhibit DNA synthesis and induce miscoding in human cells. Considering their potential role in carcinogenesis, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to explore the presence of Cro-dGuo adducts in DNA from various human tissues, such as liver, lung, and blood. DNA was isolated from human tissues and enzymatically hydrolyzed to deoxyribonucleosides. [15N 5]-Cro-dGuo was synthesized and used as an internal standard. The Cro-dGuo adducts were enriched from the hydrolysate by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring (SRM). This method allows the quantitation of the Cro-dGuo adducts at a concentration of 4 fmol/μmol dGuo, corresponding to about 1 adduct per 109 normal nucleosides starting with 1 mg of DNA, with high accuracy and precision. DNA from human liver, lung, and blood was analyzed. The Cro-dGuo adducts were detected more frequently in human lung DNA than in liver DNA but were not detected in DNA from blood. The results of this study provide quantified data on Cro-dGuo adducts in human tissues. The higher frequency of Cro-dGuo in lung DNA than in the other tissues investigated is potentially important and deserves further study.