We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E. coli DNA polymerase I (Kf) (Dosanjh et al., 1993). Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed. In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms. Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine. It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T. With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T. When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present. In contrast, Kf dissociated and re-associated rapidly. Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7. This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.