N4-Methoxydeoxycytidine 5'-triphosphate (mo4dCTP) was synthesized by reaction of dCTP with methoxyamine and then purified by high-performance liquid chromatography (HPLC) and used to analyze the specificity of mo4dCMP incorporation during polymerization on natural templates, catalyzed by DNA polymerase I of Escherichia coli. Elongation of synthetic 5'-32P-labeled primers, annealed to single-stranded DNA of bacteriophage M13, was carried out in the presence of only three of the four normal dNTPs; then, reaction products were displayed by high-resolution gel electrophoresis and visualized by autoradiography. By measuring primer elongation in each of the four "minus" reactions with and without added mo4dCTP, we examined the specificity of mo4dCMP incorporation at different positions along the M13 template. The results of this experimental approach indicated that (i) mo4dCTP is utilized most readily (although at low efficiency) in place of dTTP during DNA synthesis, (ii) the analogue can also replace dCTP during primer elongation, although at barely detectable efficiency, and (iii) the ease at which both mo4C.A and mo4C.G pairs are formed during DNA synthesis on natural templates is markedly influenced by the nucleotide sequence of the template.