A series of site-specifically modified oligodeoxynucleotides were synthesized that contained either of the two known mitomycin C-DNA monoadducts. In vitro DNA synthesis was carried out on some of these templates using a modified bacteriophage T7 DNA polymerase (Sequenase), AMV reverse transcriptase, and two different varieties of Escherichia coli DNA polymerase I (Klenow fragment)--one that carries the normal 3'-->5' exonuclease activity and a mutant protein that lacks this enzymatic function. Regardless of the type of DNA polymerase being used, DNA synthesis was terminated nearly quantitatively at the nucleotide 3' to each of these two monoadduct sites, although primer extension to full length of the template was noted with the unmodified control template. Substitution of Mn2+ for Mg2+ at a high concentration of the deoxynucleotide triphosphates resulted in incorporation of nucleotides opposite the adduct in the incubations with Sequenase or the 3'-->5' exonuclease-free Klenow fragment; however, primer extension beyond the adduct site did not take place. These studies demonstrated that the mitomycin monoadducts are strong blocks of replication and are likely to be toxic lesions in vivo. Since previous molecular modeling studies and molecular mechanical calculations indicated that the mitomycin adduction does not induce severe distortions at the site of adduction, a lack of base-pairing ability of the modified base in the extended product is unlikely to be the reason for the inhibitory effect. Instead, energy-minimized structural models indicated that additional hydrogen-bonding interactions have been introduced by the mitomycin moiety, and perhaps this increased thermodynamic stabilization of a distorted structure of the replication fork, in turn, may block the replication bypass. Experimental evidence of increased thermodynamic stability was provided by thermal melting of a template/primer complex that presumably a polymerase encounters in a typical replication fork. Consistently higher Tm of the adducted "replication fork" was noted when compared to its unmodified counterpart.