The most frequently observed mutations in ras oncogenes in solid human tumors are GCAT transitions at the 3' G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3' G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki- and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3' G compared to the 5' G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3' G and the 5' G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5' G and 3' G residues had an effect on the extent of dT misincorporation opposite each other. The 5' G was shown to have a stimulatory effect on dT misincorporation opposite the 3' G, while the 3' G was shown to have an inhibitory effect on dT misincorporation opposite the 5' G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3' G and 5' G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of human ras oncogenes, by causing GCAT transitions preferentially at the 3' G of the GG doublet in codon 12 of these oncogenes.