The ability of Klenow polymerase I, phage T7 polymerase (Sequenase®), human polymerase α, and human polymerase β to synthesize past (bypass) 06-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3′ end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single Cfi-meG in place of one G in the sequence -GlG2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before 06-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past (AmeG by DNA polymerase a and Sequen-ase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase P was completely blocked (> 2% full length extension) with 06-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase α but not Sequenase® or DNA polymerase β. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (06-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6 meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.