Methylglyoxal (MG) is a highly reactive electrophile produced endogenously as a byproduct of glucose metabolism and protein catabolism and exogenously as a food contaminant. MG reacts spontaneously with proteins, lipids, and nucleic acids to form advanced glycation end products (AGEs), modifying or inhibiting their function. Protein AGEs are associated with pathological complications of diabetes, cancer, and neurodegenerative diseases, while the physiological impact of DNA, RNA, and lipid AGE formation is less well explored. Conflicting reports in the literature on the biologically significant DNA-AGE product distribution and mechanisms of formation prompted a re-examination of the reaction products of MG with dG, oligonucleotides, and plasmid DNA under varying conditions of MG:dG stoichiometry, pH, and reaction time. Major products identified using sequential mass fragmentation and authentic standards were N2-(1-carboxyethyl)-2'-dG (CEdG), N2-(1-carboxyethyl)-7-1-hydroxy-2-oxopropyl-dG (MG-CEdG), and 1,N2-(1,2-dihydroxy-2-methyl)ethano-2'-dG (cMG-dG). CEdG and MG-CEdG were observed in all DNA substrates, although cMG-dG was not detected to any significant extent in oligomeric or polymeric DNA. Product analyses of reactions under conditions of diminished water activity as well as results from H218O labeling indicated that MG hydration equilibria plays an important role in controlling product distribution. In contrast to previous reports, our data support independent mechanisms of formation of CEdG and cMG-dG, with the latter kinetic product undergoing reversible formation under physiological conditions.