Betel nut (BN), a natural carcinogen to humans, is used as a masticator across the globe by a large segment of the human population. The primary carcinogens of BN are alkaloids, in particular arecoline. Upon nitrosation, arecoline can potentially interact with DNA, forming adducts and initiating carcinogenesis. Though considerable evidence exists in support of the carcinogenicity of BN, the molecular mechanism of its induction of carcinogenesis is unknown. This investigation was undertaken to directly demonstrate adduct formation on DNA and to study its characteristics, such as its frequency of formation and stability. A plasmid DNA construct, pMTa4, was chosen to determine the stoichiometry and dynamics of adduct formation. This construct was exposed to aqueous extract of betel nut (AEBN) in vitro and in vivo and analyzed. Spectrophotometric analysis revealed a significant red shift in the pMTa4 DNA spectrum. The gel electrophoretic mobility of pMTa4 DNA was also retarded in an AEBN dose- and exposure time-dependent manner, indicating BN-specific adduct formation on the DNA. These results conclusively demonstrate that adducts are formed on DNA by BN extract, and suggest that one AEBN-induced adduct was formed every 3 NT on pMTa4 DNA under the experimental conditions. Trace amounts of monovalent cations, such as Na+ or K+ ion (≥0.5 mmol), conferred stability to the adducts on DNA, which were otherwise unstable beyond 24 h.