DNA damage may alter the outcome of protein-nucleic acid interactions. The malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10-(3H)-one (M(1)dG), miscodes in vivo and in vitro. M(1)dG is an exocyclic adduct that undergoes ring-opening in duplex DNA to form the acyclic adduct, N(2)-(3-oxo-1-propenyl)-deoxyguanosine (N(2)-OPdG). These two adducts have different effects on DNA polymerase bypass and may affect other DNA processing enzymes. We employed the EcoRI restriction endonuclease as a model for the interaction of DNA binding proteins with adducted DNA substrates. The presence of M(1)dG in the EcoRI recognition sequence impaired the ability of the enzyme to cleave DNA, resulting in only 60% cleavage of the adducted strand and 75% cleavage of the complementary strand. Three adducts of similar structure to M(1)dG that are unable to ring-open were cleaved poorly, or not at all, by EcoRI. None of the adducts appeared to inactivate or sequester EcoRI. Additional studies with BssHII and PauI confirmed these results and demonstrated a positional effect of M(1)dG on cleavage efficiency. These data suggest dissimilar modes of protein-nucleic acid interactions based on differences in adduct structure. Comparison of the solution structures of DNA adducts and the crystal structure of EcoRI complexed to substrate suggest a model to explain the functional differences.