O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.