Methylglyoxal, a known endogenous and environmental mutagen, is a reactive alpha-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a 'primed template' DNA and the exonuclease-deficient Klenow fragment (KF(exo-)) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS-PAGE, we found that methylglyoxal induced a DNA-KF(exo-) crosslink. The specific binding complex of KF(exo-) and 'primed template' DNA was necessary for formation of the DNA-KF(exo-) crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2'-deoxyguanosine was incubated with Nalpha-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA-DNA polymerase crosslink.